Filarial nematode vaccines, polypeptides, and nucleic acids

ABSTRACT

The present invention relates to vaccines comprising a ShK domain of a filarial nematode protein. These vaccines may be used for the prevention and/or treatment of filarial nematode infections. The invention also relates to novel proteins comprising a ShK domain of a filarial nematode protein and pharmaceutical compositions. The invention may be used for the prevention and/or treatment of filarial nematode infections in canine subjects, and also in human subjects.

PRIORITY CLAIM

This application is a continuation of U.S. application Ser. No.15/118,043, now issued as U.S. Pat. No. 9,994,624, filed Aug. 10, 2016,which is a U.S. National Phase filing of PCT/GB2015/050380, filed Feb.11, 2015, which claims the benefit of British Application No. GB1402352.7, filed Feb. 11, 2014, the entire disclosures of all of whichare hereby expressly incorporated by reference herein.

FIELD OF THE INVENTION

The present invention relates to vaccines for the prevention and/ortreatment of filarial nematode infections, and to methods of preventionand/or treatment using such vaccines. The invention also relates tonovel proteins, suitable for use in the prevention and/or treatment offilarial nematode infections. The invention further relates topharmaceutical compositions comprising proteins of the invention, ornucleic acids encoding such proteins. The various aspects of the presentinvention are applicable to the prevention and/or treatment of filarialnematode infections in canine subjects, and also in human subjects.

BACKGROUND OF THE INVENTION

Nematodes are frequent infectious agents of both human and veterinaryanimal subjects. Filarial nematodes (belonging to the superfamilyFilarioidea) are responsible for a global health burden of approximately6.3 million disability-adjusted life-years, which represents thegreatest single component of morbidity attributable to helminthsaffecting humans. No vaccine exists for the major filarial diseases,lymphatic filariasis and onchocerciasis; in part because research onprotective immunity against filariae has been hampered by the inabilityof the human-parasitic species to complete their lifecycles inlaboratory mice. However, the rodent filaria Litomosoides sigmodontishas become a popular experimental model over the past two decades, asBALB/c mice are fully permissive for its development and reproduction.

Lymphatic filariasis (LF) or “elephantiasis”, which is distributedacross Africa, South Asia, the Pacific, Latin America and the Caribbean,accounts for 92% of this toll; while the remainder is caused byonchocerciasis or “river blindness”, primarily in sub-Saharan Africa.The major human filarial pathogens are Wuchereria bancrofti (which isresponsible for 90% of LF cases), Brugia malayi and Brugia timori(geographically restricted causes of LF), and Onchocerca volvulus (thesole agent of human onchocerciasis). In addition, Loa loa affects ˜13million people in West and Central Africa, generally causing arelatively mild disease, although infection has been associated withsevere and sometimes fatal adverse events following chemotherapy.Filarial parasites are primarily drivers of chronic morbidity, whichmanifests as disabling swelling of the legs, genitals and breasts in LF;or visual impairment and severe dermatitis in onchocerciasis.Furthermore, filarial parasites are also a major problem in small animalveterinary medicine, with ˜0.5 million dogs in the USA alone infectedwith Dirofilaria immitis, the cause of potentially fatal heartwormdisease.

Currently, control of human filarial diseases is almost entirelydependent on three anthelminthic drugs (ivermectin, diethylcarbamazineand albendazole), while prevention of heartworm also relies onprophylactic treatment of dogs and cats with ivermectin or othermacrocyclic lactones. Reports of potential ivermectin resistance in O.volvulus and D. immitis have highlighted the importance of maintainingresearch efforts in vaccine development against filarial nematodes.However, rational vaccine design has been constrained for severaldecades by the intrinsic complexity of these metazoan parasites andtheir multistage lifecycle, which involves uptake of the first-stagelarvae (microfilariae, Mf) by an haematophagous arthropod, two moults inthe vector, transmission of third-stage larvae (L3) to a new vertebratehost, and two further moults before the worms mature as dioecious adultsin a species-specific, parenteral predilection site. Moreover, thepresence of obligate bacterial endosymbionts (Wolbachia) in many speciesof filarial nematodes adds another level of immunogenic stimuli to thesepathogens, the impact of which remains incompletely defined. Followingthe publication of annotated genome sequences for B. malayi, D. immitisand L. loa, our understanding of the protein repertoire in filarialnematodes has been extended considerably by proteomic analyses of bothwhole body extracts (WBE) and excretory-secretory products (ESP),although only two studies (both of B. malayi) have examinedstage-specific filarial secretomes to date. In the context of vaccinedesign, the identification of ESP proteins and determination of theirexpression in each major lifecycle stage can facilitate theprioritisation of candidates for efficacy screening in animal models.

One of the most popular rodent models for filarial research, which wasfirst used during the 1940s in its natural host (the cotton rat,Sigmodon hispidus), is Litomosoides sigmodontis which was previouslydesignated as L. carinii, though this nomenclature is taxonomicallyincorrect. The utility of this model for both basic immunologicalstudies and vaccine screening changed radically with the discovery thatunlike B. malayi and indeed all other filarial species, L. sigmodontiscan complete its lifecycle in immunocompetent laboratory mice.Consequently, over the past two decades this model has drawn on the fullpower of murine immunology, including defined knockout strains, toaddress questions regarding the fundamental immunomodulatory mechanismsemployed by filarial parasites, their susceptibility to different modesof vaccination, and most recently, their ability to mitigateproinflammatory pathology and autoimmune disease. In particular, the L.sigmodontis model has been central in defining the role of T-regulatorycells in filarial immune evasion, and has enabled the assessment of theimpact of various vaccine strategies not only on adult worm burden, buton fecundity as determined by the density of Mf circulating in thebloodstream.

SUMMARY OF THE INVENTION

In a first aspect, the invention provides a polypeptide comprising a ShKdomain of a filarial nematode protein, or a variant thereof, for use asa vaccine for the prevention and/or treatment of a filarial nematodeinfection.

In a second aspect, the invention provides an artificial polypeptidecomprising a plurality of ShK domains of a filarial nematode protein, orvariants of such domains, and an artificial spacer separating the ShKdomains or variants.

In a third aspect, the invention provides a nucleic acid encoding apolypeptide according to the second aspect of the invention.

In a fourth aspect, the invention provides a nucleic acid encoding apolypeptide comprising a ShK domain of a filarial nematode protein, or avariant thereof, for use as a vaccine for the prevention and/ortreatment of a filarial nematode infection.

In a fifth aspect, the invention provides a pharmaceutical compositioncomprising a polypeptide that comprises a ShK domain of a filarialnematode protein, or a variant thereof.

In a sixth aspect, the invention provides pharmaceutical compositioncomprising a nucleic acid encoding a polypeptide that comprises a ShKdomain of a filarial nematode protein, or a variant thereof.

In a seventh aspect, the invention provides a method of preventingand/or treating a filarial nematode infection, the method comprisingproviding to a subject in need of such prevention and/or treatment atherapeutically effective amount of a polypeptide comprising a ShKdomain of a filarial nematode protein, or a variant thereof.

These various aspects of the present invention arise from the inventors'finding that polypeptides containing ShK domains from filarialnematodes, or nucleic acids encoding such polypeptides, are able toconfer protective immunity in respect of filarial nematodes associatedwith the development of diseases.

As discussed below, the various aspects of the invention have utility inthe prevention and/or treatment of filarial nematode infections in humansubjects, or in veterinary subjects such as dogs.

DETAILED DESCRIPTION OF THE INVENTION Definitions

For the avoidance of doubt, definitions will now be provided in respectof certain terms used in the description of the present invention.

“ShK Domain of a Filarial Nematode Protein”

Filarial nematodes (those that belong to the superfamily Filarioidea)are those most commonly responsible for diseases in humans, and to alesser extent, other animal hosts. A good deal of information isavailable regarding the proteome of filarial nematodes.

ShK domains, which are so called due to their similarity to theStichodactyla toxin produced by the sea anemone Stichodactylahelianthus, contain six cysteine residues with a characteristic spacing.ShK domains present in an amino acid sequence are readily identifiedusing a bioinformatics approach. For example, they are defined in thePfam database by the identifier “PF01549” and in the InterPro databaseby the identifier “IPR003582”.

The inventors have found that proteins from filarial nematode speciesthat vary quite significantly in terms of their sequence across theprotein as a whole share notably higher levels of similarity in theirShK domains. This opens the possibility of using polypeptides comprisingShK domains (or variants thereof) derived from a first filarial nematodepathogen in the prevention and/or treatment of diseases caused byinfection with a second, different, filarial nematode pathogen.

ShK domains in filarial nematode proteins are illustrated in thesequence information and comparison section of this specification. Herethe characteristic arrangement of six cysteines within the ShK domainscan be seen, as can the increased degree of sequence identity within ShKdomains of different nematodes (as compared to sequence identity sharedby the proteins as a whole).

As discussed elsewhere in the specification, the L. sigmodontis ShKdomain protein nLs_04059 represents an example of a filarial nematodeprotein; a ShK domain of which may be employed in the various aspects ofthe invention. ShK domains from this protein or orthologues of thisprotein, or variants thereof, may be employed in the various aspects ofthe invention.

nLs_04059 orthologues including suitable ShK domains, or variantsthereof, are shown in FIG. 12. These include proteins derived fromfilarial nematodes such as: L. sigmodontis (for example isoformnLS.2.1.2.t04059 (Gene ID nLs.2.1.2.g04059 Species Litomosoidessigmodontis (PRJEB3075) Location nLs.2.1.scaf00244:12218-16228), B.malayi (for example isoforms Bm8157b (Gene ID WBGene00228418, SpeciesBrugia malayi (PRJNA10729), Location Bmal_v3_scaffold110:112156-116304),Bm8157d (Gene ID WBGene00228418 Species Brugia malayi (PRJNA10729)Location Bmal_v3_scaffold110:112156-116304), Bm8157c (Gene IDWBGene00228418, Species Brugia malayi (PRJNA10729) LocationBmal_v3_scaffold110:112156-116304) and Bm12896a), A. viteae (for exampleisoform nAV.1.0.1.t09742, (Gene ID nAv.1.0.1.g09742, SpeciesAcanthocheilonema viteae (PRJEB4306) LocationnAv.1.0.scaf00135:58565-62790), D. immitis (for example isoformsnDi.2.2.2.t03402 (Gene ID nDi.2.2.2.g03402 Species Dirofilaria immitis(PRJEB1797) Location nDi.2.2.scaf00051:142071-145588), andnDi.2.2.2.t04314 (Gene ID nDi.2.2.2.g04314, Species Dirofilaria immitis(PRJEB1797) Location nDi.2.2.scaf00083:48251-52806), L. loa (for exampleisoforms EJD74930.1 (Gene ID LOAG_17825, Species Loa loa (PRJNA60051)Location JH712199:22673-23334), and EJD74931.1 (Gene ID LOAG_17826,Species Loa loa (PRJNA60051) Location JH712199:24313-26569), O. ochengi(for example isoforms nOo.2.0.1.t12220 (Gene ID nOo.2.0.1.g12220 SpeciesOnchocerca ochengi (PRJEB1809) Location nOo.2.0.Scaf09993:244-1668),nOo.2.0.1.t06172 (Gene ID nOo.2.0.1.g06172, Species Onchocerca ochengi(PRJEB1809) Location nOo. 2.0. Scaf01844:10830-11939), andnOo.2.0.1.t06343 (Gene ID nOo.2.0.1.g06343, Species Onchocerca ochengi(PRJEB1809) Location nOo.2.0.Scaf01943:3177-7069). O. volvulus (forexample isoforms OVOC 0000232701.1, and OVOC 000102301.1) and W.bancrofti (for example isoforms WUBG 17834T0, and WUBG05152T0).

All Gene ID sequences identified in the preceding paragraph are fromWormBase ParaSite database version 1 (September 2014).

For the sake of brevity, the specification will use the terms “ShKdomain of a filarial nematode protein” and “Shk domain” interchangeably.

“Variants” of Domains

For the purposes of the present disclosure, a variant of a ShK domain ofa filarial nematode protein should be considered to encompass sequencessharing at least 70% identity with a ShK domain of a filarial nematodeprotein. Optionally, variants may share at least 75% identity, at least80% identity, at least 85% identity, or at least 90% identity with a ShKprotein. In suitable embodiments, variants may share at least 91%identity, at least 92% identity, at least 93% identity, at least 94%identity, at least 95% identity, at least 96% identity, at least 97%identity, at least 98% identity, at least 99% identity with a Shk domainof a filarial nematode protein.

It should be borne in mind that the overall level of identity between L.sigmodontis ShK domain protein nLs_04059 (SEQ ID NO: 1) and itsorthologues in filarial pathogens ranges from 31% (in W. bancrofti) to51% (in O. volvulus). However, the level of identity between individualShK domains between species always exceeds 70% and preferably exceeds85%,

It will be appreciated that, in order to function as a useful vaccine, apolypeptide of the invention should exhibit the ability to induce aprotective immune response. Suitably a variant may retain at least 70%of the immunogenic capacity of the ShK domain from which it is derived.Indeed, a variant may retain at least 80%, at least 90%, or even atleast 95% of the immunogenic capacity of the ShK domain from which it isderived. Suitably a variant may have a greater immunogenic capacity thanthe ShK domain from which it is derived.

“Prevention and/or Treatment”

The medical uses, methods of treatment, and pharmaceutical compositionsof the invention may be used to establish protective immunity thatprevents the establishment of a filarial nematode infection in asubject. This prophylactic use exemplifies the “prevention” of afilarial nematode infection as this term is used in the presentdisclosure.

The advantages of these various aspects of the invention may also beapplicable to subjects that have previously undergone infection with afilarial nematode parasite. For the purposes of the present disclosure,such applications of the invention, in which a disease associated withan existing infection is alleviated, may be considered to represent“treatment” of the filarial nematode infection.

Prevention and/or treatment of a filarial nematode infection bringsabout a corresponding prevention and/or treatment of the diseaseassociated with the filarial nematode infection.

“Polypeptides of the Invention”

In the context of the present disclosure, references to “a polypeptideof the invention” or to “polypeptides of the invention” should be takenas encompassing not only the artificial polypeptides of the secondaspect of the invention, but also the polypeptides for medical use (asvaccines for the prevention and/or treatment of a filarial nematodeinfection) defined by the first aspect of the invention.

As discussed further below, the medical uses of the first aspect of theinvention may employ naturally occurring polypeptides, or may make useof artificial polypeptides such as those of the second aspect of theinvention.

For the sake of brevity, the majority of the following embodiments ofthe invention will be discussed primarily in the context of polypeptidesof the invention, whether the polypeptides for medical use of the firstaspect of the invention, or the artificial polypeptides of the secondaspect of the invention. However, it should be appreciated that theconsiderations set out herein in respect of polypeptides of theinvention will also, except for where the context requires otherwise, beapplicable to the other aspects of the invention, such as nucleic acids(where the considerations may be applicable to the polypeptides encodedby such nucleic acids sequences), pharmaceutical compositions, andmethods of treatment.

As referred to above, the polypeptide or nucleic acids of the inventionare suitable for use as vaccines, where the vaccine is for theprevention and/or treatment of a filarial nematode infection.

In a suitable embodiment, a polypeptide of the invention may comprise anShK domain from L. sigmodontis. As the results set out hereinillustrate, the inventors have believe that polypeptides comprising ShKdomains from L. sigmodontis (and specifically nucleic acids encodingsuch polypeptides) are surprisingly able to act as vaccines conferringprotective immunity in respect of infections by filarial nematodes otherthan L. sigmodontis.

Without detracting from the above, in a suitable embodiment apolypeptide in accordance with the invention comprises an ShK domainfrom a filarial nematode infection which is to be prevented and/ortreated (or a variant of such an ShK domain).

Suitably, a polypeptide for of the invention may be for use in theprevention and/or treatment of canine heartworm. In such an embodiment,the polypeptide may comprise a ShK domain from D. immitis, or a variantthereof.

A polypeptide of the invention may be for use in the prevention and/ortreatment of a disease in a human subject, the disease being selectedfrom the group consisting of: lymphatic filariasis (also referred to as“elephantiasis”); onchocerciasis (also referred to as “riverblindness”); and loiasis.

Suitably a polypeptide of the invention for use in the prevention and/ortreatment of lymphatic filariasis will comprise a ShK domain from afilarial nematode selected from the group consisting of: Wuchereriabancrofti; Brugia malayi; and Brugia timori, or a variant thereof. Ofthese three filarial nematodes, W. bancrofti is responsible forapproximately 90% of lymphatic filariasis cases, and so polypeptidescomprising an ShK domain from W. bancrofti may be preferred for use inthe prevention and/or treatment of lymphatic filariasis.

In an embodiment in which a polypeptide of the invention is for use inthe prevention and/or treatment of onchocerciasis, it may comprise a ShKdomain from Onchocerca volvulus, or a variant thereof.

A suitable polypeptide for use in the prevention and/or treatment ofloiasis, may comprise a ShK domain from Loa loa, or a variant thereof.

In a suitable embodiment, a polypeptide of the invention comprises aplurality of ShK domains, or variants thereof. Thus, by way ofnon-limiting example, a polypeptide of the invention may comprise atleast two, at least three, at least four, at least five, or at least sixShK domains or variants thereof. In suitable embodiments, a polypeptideof the invention may comprise two, three, four, five or six ShK domainsor variants thereof.

In the event that a polypeptide according to the invention comprises aplurality of ShK domains (or variants thereof), it may comprise aplurality of the same ShK domain (or variants of the same ShK domain).In an embodiment utilising variants of the same ShK domain these variantmay be the same variant, or may comprise a plurality of differentvariants.

Alternatively, a polypeptide of the invention comprising a plurality ofShK domains (or variants thereof), it may comprise a plurality of thedifferent ShK domains (or variants of these different domains). Suitablyeach one of the plurality of ShK domains may be different, oralternatively the polypeptide may comprise more than one copy of asingle ShK domain among a plurality of different domains.

Merely by way of example, in the case of D. immitis the naturallyoccurring ShK domain protein contains six ShK domains, each of which hasits own characteristic sequence. A polypeptide of the invention maycomprise each of these six ShK domains. Alternatively, a polypeptide ofthe invention may comprise six ShK domains made up of six copies of thesame ShK sequence, such as the sixth of the sequences found in thenative protein.

The sixth ShK sequence found in the native ShK domain protein of D.immitis may represent a preferred ShK domain to be included (eitherdirectly, or in variant form) in a polypeptide of the invention. Thus,in a suitable embodiment a polypeptide of the invention may comprise oneor more ShK domains (or variants thereof) selected from ShK domains oneto five of the native protein, in addition to the sixth ShK domain (or avariant thereof).

In the event that a polypeptide of the invention comprises only a singleShK domain derived from D. immitis, the single ShK domain may be thesixth ShK domain from the ShK domain protein of D. immitis (or a variantbased upon this domain).

It will be appreciated that the considerations set out in the precedingparagraphs also apply to polypeptides of the invention comprisingvariants of the ShK domains found in D. immitis.

A polypeptide of the invention may be a branched protein. In a suitableembodiment, each branch of the protein may carry an antigenic sequence.Some, and potentially all, of these antigenic sequences may comprise ShKdomains, or their variants.

In a suitable embodiment, a polypeptide of the invention may furthercomprise an additional antigen that is able to confer protectiveimmunity on a subject to whom the additional antigen is provided.Suitably such a polypeptide may comprise an additional antigen that doesnot comprise an ShK domain.

In a suitable embodiment, the additional antigen incorporated in such apolypeptide may be a further nematode antigen. Suitably the additionalantigen capable of conferring protective immunity is derived from thesame filarial nematode as the ShK domain incorporated in thepolypeptide.

Some of the embodiments referred to above may be provided by naturallyoccurring polypeptides comprising an ShK domain. In a suitableembodiment a polypeptide to be employed in accordance with the variousaspects or embodiments of the invention may be a naturally occurringpolypeptide.

Certain of the embodiments referred to above may only be provided byartificial polypeptides comprising an ShK domain. As set out above, thesecond aspect of the invention provides an artificial polypeptidecomprising a plurality of ShK domains of a filarial nematode protein, orvariants thereof, and an artificial spacer separating the ShK domains orvariants.

A suitable artificial spacer serves to expose the ShK domains to cellsof the immune system, thereby allowing the development of protectiveimmunity. The spacer itself need not contribute to the development ofthe protective immunity and may itself be immunologically inert.

The artificial spacer may be any spacer, other than naturally occurringsequence found between ShK domains in a natural protein, that serves toseparate the ShK domains, or variants, within the artificial protein. Ina suitable embodiment an artificial spacer suitable for use in theartificial polypeptides of the invention may comprise a sequence ofamino acid residues that separates the ShK domains or variant. In asuitable embodiment the spacer may comprise poly-L-lysine.

Artificial polypeptides of the invention may comprise a plurality ofartificial spacers, as necessitated by the number of ShK domains (orvariants thereof) incorporated in the artificial polypeptide.

In a suitable embodiment a polypeptide to be employed in accordance withthe various aspects or embodiments of the invention may be an artificialpolypeptide, such as an artificial polypeptide of the 2^(nd) aspect ofthe invention.

Suitably an artificial polypeptide of the invention may be a chimericpolypeptide. Artificial polypeptides of the invention may comprise a ShKdomain, or variant thereof, and an additional antigen that is not foundin the polypeptide from which the ShK domain is derived. Merely by wayof example, an artificial protein of the invention may comprise an ShKdomain (or variant thereof) and an additional antigen from a nematodethat the ShK domain is derived from, or an additional antigen from anematode other than that which the ShK is derived from, or an additionalantigen that is derived from a source other than a nematode. Chimericpolypeptides of the invention comprising an ShK domain or variantthereof, and an additional antigen from a source other than the filarialnematode from which the ShK domain was derived are able to induceprotective immunity against more than one pathogen.

Artificial polypeptides of the invention may comprise a plurality of thesame ShK domain, or variants thereof. Alternatively, artificialpolypeptides of the invention may comprise a plurality of different ShKdomains, or variants thereof.

In a suitable embodiment an artificial polypeptide of the inventionfurther comprises an additional vaccine antigen. Suitably the additionalvaccine antigen may be derived from an antigen that does not comprise aShK domain.

An artificial polypeptide of the invention may comprise an additionalvaccine antigen derived from the same filarial nematode (or filarialnematodes) as the ShK domains incorporated in the polypeptide.

Examples of suitable additional vaccine antigens that may beincorporated in artificial polypeptides of the invention includecysteine proteinase inhibitor (CPI) and/or abundant larval transcript(ALT). As discussed elsewhere in the specification, these proteinsrepresent secreted immunomodulators secreted by female filarialnematodes, and targeting of these immunomodulators by vaccination leadsto greatly reduced microfilaremial. Accordingly, introduction of theseadditional vaccine antigens into artificial polypeptides of theinvention will be expected to confer therapeutic advantages that gobeyond the surprising benefits provided by the polypeptides of theinvention. Therapeutic vaccination of Onchocerca volvulus-infected hostswith vaccines comprising CPI and/or ALT in combination with the ShKdomain-containing polypeptides of the invention may provide furthersuppression of microfilarial production, prevent the progression ofdisease, reduce morbidity and block transmission, even if adult wormburden remains unaffected.

Furthermore, it will be appreciated that in a suitable embodiment apolypeptide, medical use, or method of treatment of the inventionutilising as a vaccine a polypeptide comprising a ShK domain of afilarial nematode protein, or a variant thereof, may be used inconjunction with a vaccine comprising CPI and/or ALT. Suitably thepolypeptide of the invention may be provided in the same vaccine as theCPI and/or ALT. Alternatively the polypeptide of the invention and theCPI and/or ALT may be provided in separate vaccines.

The invention provides a nucleic acid encoding a polypeptide of theinvention. The nucleic acid may encode an artificial polypeptide inaccordance with the second aspect of the invention.

The invention also provides a vector comprising a nucleic acid of theinvention, and such a vector may be adapted for expression in bacteria,such as E. coli.

Pharmaceutical compositions of the invention may comprise a polypeptideof the invention and/or a nucleic acid of the invention. The nucleicacid may be provided in the form of a vector.

Suitable pharmaceutical compositions of the invention may be formulatedfor use as a vaccine, and may be formulated for any appropriate route ofadministration, including (but not limited to) injection.

That said, suitable routes of administration include, but are notlimited to, oral (e.g, by ingestion); buccal; sublingual; transdermal(including, e.g., by a patch, plaster, etc.); transmucosal (including,e.g., by a patch, plaster, etc.); intranasal (e.g., by nasal spray);ocular (e.g., by eyedrops); pulmonary (e.g., by inhalation orinsufflation therapy using, e.g., via an aerosol, e.g., through themouth or nose); rectal (e.g., by suppository or enema); vaginal (e.g.,by pessary); parenteral, for example, by injection, includingsubcutaneous, intradermal, intramuscular, intravenous, intraarterial,intracardiac, intrathecal, intraspinal, intracapsular, subcapsular,intraorbital, intraperitoneal, intratracheal, subcuticular,intraarticular, subarachnoid, and intrasternal; by implant of a depot orreservoir, for example, subcutaneously or intramuscularly.

Pharmaceutical compositions in accordance with the invention may beformulated such that the polypeptide or nucleic acid of the invention isdelivered in alum adjuvant, or in virus-like particles.

As referred to above, the seventh aspect of the invention provides amethod of preventing and/or treating a filarial nematode infection, themethod comprising providing to a subject in need of such preventionand/or treatment a therapeutically effective amount of a polypeptidecomprising a ShK domain of a filarial nematode protein, or a variantthereof. For brevity, such methods may be referred to in the presentdisclosure as “methods of treatment”, but, unless the context requiresotherwise, it should be considered that such methods of treatment alsoencompass prophylactic use to prevent filarial nematode infections.

It will be appreciated that the various polypeptides of the inventiondescribed herein represent suitable polypeptide to be used in suchmethods of treatment, and the various considerations set out inconnection with the nature of such polypeptides will also be applicableto polypeptides for use in such methods of treatment.

The methods of treatment of the invention are applicable to veterinarysubjects. In a suitable embodiment of a method of the invention thesubject is a dog, and the filarial nematode infection to be preventedand/or treated is heartworm. In a method in which it is desired toprevent and/or treat heartworm, the polypeptide provided in the methodsuitably comprises a ShK domain from D. immitis, or a variant thereof.

The methods of treatment of the invention are also applicable to humansubjects. Suitably, when the subject is a human, the filarial nematodeinfection to be prevented and/or treated is one that causes a diseaseselected from the group consisting of: lymphatic filariasis;onchocerciasis; and loiasis.

In an embodiment of the invention in which the disease to be preventedand/or treated is lymphatic filariasis, the polypeptide suitablycomprises a ShK domain from Wuchereria bancrofti; Brugia malayi; andBrugia timori, or a variant thereof.

W. bancrofti is responsible for approximately 90% of lymphaticfilariasis cases, and so it may be preferred that methods for theprevention and/or treatment of lymphatic filariasis make use ofpolypeptides comprising an ShK domain from W. bancrofti.

Suitably a method of the invention in which it is wished to preventand/or treat onchocerciasis may make use of a polypeptide comprising aShK domain from Onchocerca volvulus, or a variant thereof.

Methods of the invention in which it is desired to prevent and/or treatloiasis may make use of a polypeptide that comprises a ShK domain fromLoa loa, or a variant of such a domain.

The skilled reader will appreciate that in suitable embodiments ofmethods of treatment in accordance with the invention, thetherapeutically effective amount of the polypeptide is provided byadministration of the polypeptide. The therapeutically effective amountmay be provided through single or multiple incidences of administration,as required. Suitably such embodiments of the invention may utilisepharmaceutical compositions of the invention for the provision of therequired amount of the polypeptide.

The invention also encompasses methods of treatment in which thetherapeutically effective amount of the polypeptide is provided byadministration of a nucleic acid encoding the polypeptide, for exampleby provision of the nucleic acid in a suitable vector. In suchembodiments expression of the nucleic acid by the cells of the recipientsubject leads to the production of the therapeutically effective amountof the protein, thus leading to the development of protective immunity.These embodiments of the methods of treatment may utilise pharmaceuticalcompositions comprising nucleic acids, which are also aspects of thepresent invention.

Factors that may be considered in the determination of a therapeuticallyeffective amount of a polypeptide, variant, or nucleic acid of theinvention may include: the nature of the agent in question (i.e. whetherthe agent in question is a polypeptide, a variant thereof, or a nucleicacid); the activity of the agent in question; the severity of theinfection to be prevented and/or treated; the size of the subjectrequiring prevention and/or treatment; and the route by which the agentis to be administered.

Merely by way of example, a therapeutically effective amount of apolypeptide comprising a ShK domain, or a variant thereof, or a nucleicacid encoding such a polypeptide or variant, may be between 1.5 g and 1μg. A suitable therapeutically effective amount may be between 1500 mgand 1 mg; for example between 1000 mg and 50 mg; such as between 500 mgand 100 mg. Alternatively a suitable therapeutically effective amountmay be between 100 mg and 1 mg; for example between 50 mg and 5 mg; suchas between 25 mg and 10 mg. In a further suitable embodiment, a suitabletherapeutically effective amount may be between 500 μg and 1 μg; forexample between 400 μg and 5 μg; such as between 250 μg and 10 μg.Merely by way of example, a suitable therapeutically effective amountmay be between 200 μg and 15 μg, such as between 150 μg and 20 μg,between 100 μg and 25 μg, or between 50 μg and 30 μg. Suitably atherapeutically effective amount may be approximately 40 μg.

Within a course of treatment to prevent and/or treat a filarial nematodeinfection a polypeptide comprising a ShK domain, or a variant thereof,or a nucleic acid encoding such a polypeptide or variant, may beprovided in one or more administrations. Incidences of administrationmay be provided once per 24 hours, once a week, once, a month, or asotherwise required.

The invention will now be further described with reference to thefollowing Experimental Results and Figures in which:

FIG. 1A. Graph illustrating the difference in parasite burden betweenvaccinated and non-vaccinated mice in Study 1 below;

FIG. 1B. Distribution of ESP proteins between life stages of L.sigmodontis. Venn diagram of the shared and stage-specific ESP proteinsin each of the life stages examined.

FIG. 2. Pfam enrichment analysis of ESP proteins against the completetheoretical proteome of L. sigmodontis. The fold-enrichment is displayedfor each lifecycle stage; DUF290 represents the transthyretin-likeprotein family.

FIG. 3. Relative abundance of L. sigmodontis ESP proteins compared tocorresponding somatic extracts. ESP proteins (≥2 peptides detected atp<0.05 and <1% FDR, present in ≥2 biological replicates) were quantifiedby ion intensity (iBAQ) and compared to the iBAQ abundance of the sameprotein present in somatic extracts of intact nematodes (x-axis).Individual abundance values were normalised by dividing by the summedtotal abundance of that individual sample (life stage). The normalisedabundance ratio was used as a guide to evaluate the enrichment of theprotein (ESP/WBE). Note that as the data are normalised within each lifestage dataset, comparing protein abundance directly between life stagesis not valid.

FIG. 4A. Comparison of ESP protein abundance (iBAQ) in adult stages ofL. sigmodontis. The top 35 most abundant proteins in each ES preparation(adult male, “AM”) are ranked by normalised iBAQ abundance (stripedbars); the corresponding abundance in WBE is displayed for comparison(white bars) in a stacked format. Individual protein abundance valueswere normalised by the summed total abundance per sample. An asteriskindicates proteins with a predicted signal peptide, while predictedsecretion through the non-classical pathway is indicated by a plus sign.

FIG. 4B. Comparison of ESP protein abundance (iBAQ) in adult stages ofL. sigmodontis. The top 35 most abundant proteins in each ES preparation(adult gravid female, “GAF”) are ranked by normalised iBAQ abundance(striped bars); the corresponding abundance in WBE is displayed forcomparison (white bars) in a stacked format. Individual proteinabundance values were normalised by the summed total abundance persample. An asterisk indicates proteins with a predicted signal peptide,while predicted secretion through the non-classical pathway is indicatedby a plus sign.

FIG. 4C. Comparison of ESP protein abundance (iBAQ) in adult stages ofL. sigmodontis. The top 35 most abundant proteins in each ES preparation(adult pre-gravid female, “PAF”) are ranked by normalised iBAQ abundance(striped bars); the corresponding abundance in WBE is displayed forcomparison (white bars) in a stacked format. Individual proteinabundance values were normalised by the summed total abundance persample. An asterisk indicates proteins with a predicted signal peptide,while predicted secretion through the non-classical pathway is indicatedby a plus sign.

FIG. 5A. Comparison of ESP protein abundance (iBAQ) in larval stages ofL. sigmodontis. Proteins in each ESP preparation (vL3) are ranked bynormalised iBAQ abundance (striped bars); the corresponding abundance inWBE is displayed for comparison (white bars) in a stacked format.Individual protein abundance values were normalised by the summed totalabundance per sample. An asterisk indicates proteins with a predictedsignal peptide, while predicted secretion through the non-classicalpathway is indicated by a plus sign.

FIG. 5B. Comparison of ESP protein abundance (iBAQ) in larval stages ofL. sigmodontis. Proteins in each ESP preparation (iMf) are ranked bynormalised iBAQ abundance (striped bars); the corresponding abundance inWBE is displayed for comparison (white bars) in a stacked format.Individual protein abundance values were normalised by the summed totalabundance per sample. An asterisk indicates proteins with a predictedsignal peptide, while predicted secretion through the non-classicalpathway is indicated by a plus sign.

FIG. 6. The ShK domains from L. sigmodontis protein nLs_04059 and itsorthologues in other filarial species have a distinct sequencesignature. All ShK domains identified in the complete theoreticalproteomes of L. sigmodontis, B. malayi, L. loa, W. bancrofti, O.ochengi, D. immitis, Acanthocheilonema viteae and Ascaris suum wereextracted and aligned, and sequence logos derived from: (all ShK) all531 domains, (04059) all domains from nLs_04059 and its orthologues,(1-6) the aligned orthologous domains 1 to 6 from nLs_04059 and itsorthologues. No nLs_04059 orthologue was found in A. suum. As thenLs_04059 domains are relatively short, there are gaps in the sequencelogos for the nLs_04059-derived domains. Numbering in all the panels isbased on the full ShK alignment.

FIG. 7A. Number of adult ES proteins detected in published studies of B.malayi adults and comparison with orthologues present in the L.sigmodontis adult secretome. The study-specific and shared proteinsrepresent combined data from both adult sexes. Note that proteinidentifications are those quoted by each individual study andstatistical cut-offs have not been standardised. Brugia malayiorthologues of L. sigmodontis proteins were identified by reciprocalBLAST of the respective theoretical proteomes (bit score >50). Thedistribution of the orthologues in adult nematode ESP across threepreviously published studies (B. malayi) and the current study (L.sigmodontis) is displayed.

FIG. 7B. Number of adult ES proteins detected in published studies of B.malayi adults and comparison with orthologues present in the L.sigmodontis adult secretome. The study-specific and shared proteinsrepresent combined data from both adult sexes. Note that proteinidentifications are those quoted by each individual study andstatistical cut-offs have not been standardised. Brugia malayiorthologues of L. sigmodontis proteins were identified by reciprocalBLAST of the respective theoretical proteomes (bit score >50). Thedistribution of species-specific (non-orthologous) proteins issummarised.

FIG. 8. Heat-map of protein profiles for excretory-secretorypreparations and whole body extracts of Litomosoides sigmodontis.Dendrograms shown in this Figure were generated by hierarchicalclustering based on pair-wise distance. ESP, excretory-secretoryproducts; WBE, whole body extracts; GAF, gravid adult females; PAF,pre-gravid adult females; AM, adult males; iMF, immature microfilariae;vL3, vector-derived third stage larvae.

FIG. 9. Domain organisation of protein nLs_04059 from Litomosoidessigmodontis. Linear representation of the amino-acid sequencehighlighting the signal peptide (italicised), six ShK toxin-like domains(open rectangles) containing six cysteine residues each (highlighted),and a predicted propeptide cleavage site (underlined). Domain six at theC-terminus is unique in containing two lysyltyrosine dyads (bold).

FIG. 10. Amino-acid sequence alignment of L. sigmodontis proteinnLs_03577 and its orthologues in other filarial nematodes. Homologues ofnLs_03577 were identified by BLASTp search of protein databases fromsequenced nematode genomes and a transcriptome assembly for Setarialabiatopapillosa (G. Koutsovoulos, B. Makepeace, M. Blaxter;unpublished). No homologues were found outside the filarial nematodes.The protein sequences were aligned with ClustalOmega, and identity isindicated by a coloured scale (high identity is indicated in striped;lower identity is indicated in white).

FIG. 11. Rooted phylogenetic tree of L. sigmodontis protein nLs_03577and its orthologues in other filarial nematodes. Homologues of nLs_03577were identified by BLASTP search of protein databases from sequencednematode genomes and a transcriptome assembly for Setarialabiatopapillosa (G. Koutsovoulos, B. Makepeace, M. Blaxter;unpublished). No homologues were found outside the filarial nematodes.The protein sequences were aligned with ClustalOmega and the alignmentsubjected to phylogenetic analysis using MrBayes version 3.2. Every100^(th) generation from the final 1 million generations of a 2 milliongeneration analysis were combined to derive the consensus shown. Thetree is rooted with S. labiatopapillosa, in accordance with acceptedsystematics, and nuclear small subunit ribosomal RNA phylogeny.

FIG. 12. Rooted phylogenetic tree of ShK domains among predictedproteins in filarial nematodes. The rooted subtrees for the six ShKdomains from the nLs_04059 orthologues are shown. In B. malayi, domain 1is represented by two distinct isoform clusters, one of which (Bm1) isfound only in this species and in W. bancrofti.

FIG. 13. Unrooted phylogenetic tree of ShK domains among predictedproteins in filarial nematodes and Ascaris suum. The numbers 1-6identify the ShK domains of the L. sigmodontis protein nLs_04059.

FIG. 14. Distribution of biotin in labelled and unlabelled specimens ofadult Litomosoides sigmodontis. Fixed worm sections were incubated withstreptavidin-FITC. A, Biotin-labelled works. B, An unlabelled controlspecimen.

EXPERIMENTAL RESULTS

Study 1

The ability of polypeptides comprising ShK domains of filarial nematodeproteins to as vaccines conferring protective immunity in respect offilarial nematode infection, and the suitability of nucleic acidsencoding such polypeptides to serve as vaccines, was demonstrated by thefollowing study. The L. sigmodontis ShK domain containing protein usedas an exemplary vaccine was designated LsShK for the purposes of thisstudy.

Immunisations and infections were performed with female BALB/c mice,starting at ages of 6-7 weeks, with five animals per experimental group.Mice were housed in individually ventilated cages and infectedsubcutaneously with 30 or 40 L. sigmodontis infective larvae (iL3).Naïve, uninfected animals were maintained and sampled in parallel ascontrols for the immunological readouts.

All cloning was carried out following the recommendations of the pcDNA3.1 Directional TOPO Expression Kit (Invitrogen). LsShK (gene IDnLs.2.1.2.t04059-RA) was amplified from a cDNA preparation of adult L.sigmodontis using specific primers. Fusion constructs containingsingle-chain anti-DEC205 antibody (DEC) upstream of the LsShK sequencewere produced from ready-made constructs kindly provided by Dr. RalphSteinman. Briefly, PCR products of genes of interest were digested withNotI and XbaI (Neb laboratory, UK), then ligated into an NotI andXbaI-digested anti-mouse dec-205 single chain antibody-ovalbuminconstruct (DEC-OVA) or antibody control Ig-OVA to replace the fragmentof OVA gene, respectively. All plasmids were sequenced to confirmidentity.

Plasmids were injected in the tibialis anterior muscle of the left legwith a 27 G needle, immediately followed by electroporation with an ECM830 generator+Tweezertrodes (BTX Harvard Apparatus) using as settings 8pulses, 200 V/cm, 40 ms duration, 460 ms interval. Each mouse wasimmunised twice separated by 2 weeks interval with 40 μg of DNA totalmade up by equal quantities of each plasmid species, delivered in 50 μlPBS. As a consequence, the quantity of each individual plasmid wasreduced as the number of different plasmids incorporated into theinoculums increased. However, the quantity of each one remained inexcess of the minimal efficient dose.

Parasite survival was determined at experiment endpoint. Adult filariaewere isolated from the pleural cavity lavage fluid in 10 ml cold PBS,fixed in hot 70% ethanol and counted. Protection was calculated as:(mean burden in primary infected animals−mean burden of vaccinatedanimals)/mean burden in primary infected animals.

Microfilariae were counted in 30 μl of blood after fixation in 570 μl ofBD FACS lysing solution (BD Biosciences) under an inverted microscope.

Generalised linear models were used to compare the effects of differentvaccine formulations on parasitological parameters as they allow moreflexibility in specifying the distribution of response variables andbetter model fitting through Maximum Likelihood estimation.

The results of this study are illustrated in FIG. 1.

The inventors found that at day 60 post-infection, the LsShK vaccine hada modest effect on adult worm burden (˜40% reduction), though this wasof borderline statistical significance (p=0.07). However, the vaccinatedgroup had no microfilariae detected in the blood, whereas the primaryinfection group (which received empty plasmid vector only) exhibited amedian microfilaraemia of ˜830 parasites per ml (p=0.005). This suggeststhat the medical use of ShK domains as vaccines achieves its therapeuticuse through sterilising the adult female worms, or by killing migratingmicrofilariae before they can reach the bloodstream.

Study 2

The invention may further be understood by the skilled person onconsideration of the following details of a study undertakingquantitative secretome analysis of a model filarial nematode(Litomosoides sigmodontis) across the parasite life cycle.

2.1 Summary

Filarial nematodes (superfamily Filarioidea) are responsible for anannual global health burden of approximately 6.3 milliondisability-adjusted life-years, which represents the greatest singlecomponent of morbidity attributable to helminths affecting humans. Novaccine exists for the major filarial diseases, lymphatic filariasis andonchocerciasis; in part because research on protective immunity againstfilariae has been constrained because the human-parasitic species cannotcomplete their lifecycles in laboratory mice. However, the rodentfilaria Litomosoides sigmodontis has become a popular experimentalmodel, as BALB/c mice are fully permissive for its development andreproduction. Here, we provide a comprehensive analysis ofexcretory-secretory products from L. sigmodontis across five lifecyclestages. Applying intensity-based quantification, we determined theabundance of 302 unique excretory-secretory proteins, of which 64.6%were present in quantifiable amounts only from gravid adult femalenematodes. This lifecycle stage, together with immature first-stagelarvae (microfilariae), released four proteins that have not previouslybeen evaluated as vaccine candidates: a predicted 28.5 kDafilaria-specific protein, a zonadhesin and SCO-spondin-like protein, avitellogenin, and a protein containing six metridin-like ShK toxindomains. Female nematodes also released two proteins derived from theobligate Wolbachia symbiont. Notably, excretory-secretory products fromall parasite stages contained several uncharacterised members of thetransthyretin-like protein family. Furthermore, biotin labellingrevealed that redox proteins and enzymes involved in purinergicsignalling were enriched on the adult nematode cuticle. Comparison ofthe L. sigmodontis adult secretome with that of the human-infectivefilarial nematode Brugia malayi (reported previously in threeindependent published studies) identified differences that suggest aconsiderable underlying diversity of potential immunomodulators. Themolecules identified in L. sigmodontis excretory-secretory products showpromise not only for vaccination against filarial infections, but forthe amelioration of allergy and autoimmune diseases.

2.2 Introduction

Filarial nematodes are the most important helminth parasites of humansin terms of overall impact on public health, with an annual globalburden of ˜6.3 million disability-adjusted life-years (1). Lymphaticfilariasis (LF) or “elephantiasis”, which affects populations acrossAfrica, South Asia, the Pacific, Latin America and the Caribbean,accounts for 92% of this toll. The remainder is caused by onchocerciasisor “river blindness”, primarily in sub-Saharan Africa. The major humanfilarial pathogens are Wuchereria bancrofti (responsible for 90% of LFcases), Brugia malayi and Brugia timori (geographically restrictedcauses of LF), and Onchocerca volvulus (the sole agent of humanonchocerciasis). In addition, Loa loa affects ˜13 million people in Westand Central Africa. This parasite usually induces a relatively milddisease, but has been associated with severe and sometimes fatal adverseevents following anthelmintic chemotherapy (2). Filarial parasites areprimarily drivers of chronic morbidity, which manifests as disablingswelling of the legs, genitals and breasts in LF; or visual impairmentand severe dermatitis in onchocerciasis. The filariae are also a majorproblem in small animal veterinary medicine, with ˜0.5 million dogs inthe USA alone infected with Dirofilaria immitis (3), the cause ofpotentially fatal heartworm disease. However, in domesticated ungulates,filarial infections are generally quite benign (4).

Currently, control of human filarial diseases is almost entirelydependent on three drugs (ivermectin, diethylcarbamazine andalbendazole). Prevention of heartworm also relies on prophylactictreatment of dogs and cats with ivermectin or other macrocycliclactones. Reports of possible ivermectin resistance in O. volvulus (5)and D. immitis (6) have highlighted the importance of maintainingresearch efforts in vaccine development against filarial nematodes.However, rational vaccine design has been constrained for severaldecades (7) by the intrinsic complexity of these metazoan parasites andtheir multistage lifecycle. Moreover, many filarial species carryobligate bacterial endosymbionts (Wolbachia), which may also stimulatethe immune response during infection (8). As part of global efforts toimprove prevention and treatment of these diseases, large-scale projectshave been undertaken, including sequencing of the nematodes (9-11) andtheir Wolbachia (10, 12, 13), and proteomic analyses of both wholeorganisms and excretory-secretory products (ESP) (14, 15). Additionally,two studies (both on B. malayi) have examined lifecycle stage-specificsecretomes (16, 17). In the context of vaccine design, theidentification of ESP proteins and determination of their expression ineach major lifecycle stage can facilitate the prioritisation ofcandidates for efficacy screening in animal models.

One barrier to the progression of research in the filarial field is ourinability to maintain the full lifecycle of the human parasites ingenetically tractable, inbred hosts. The filarial lifecycle involvesuptake of the first-stage larvae (microfilariae, Mf) by a haematophagousarthropod, two moults in this vector, followed by transmission ofthird-stage larvae (L3) to a new vertebrate host. Two further moultsoccur in the definitive host before the nematodes mature as dioeciousadults in a species-specific, parenteral predilection site. However, thecomplete lifecycle of the New World filaria Litomosoides sigmodontis canbe maintained in laboratory rodents, including inbred mice (18). Thisspecies was first studied in its natural host (the cotton rat, Sigmodonhispidus) (19) [the previous designation of these isolates as L. cariniiis taxonomically incorrect (20)]. Drawing on the full power of murineimmunology, including defined knockout strains, this model has beenaddress questions regarding the fundamental immunomodulatory mechanismsemployed by filarial parasites (21), their susceptibility to differentmodes of vaccination, their ability to mitigate proinflammatorypathology and autoimmune disease (22), and the impact of various vaccinestrategies on adult nematode burden and fecundity (23) (24). The L.sigmodontis model has also been central in defining the role ofT-regulatory cells in filarial immune evasion (25).

Using the resource of a newly-determined genome sequence, coupled with aderivative of intensity-based absolute quantification (iBAQ) proteomics,we have examined the stage-specific secretome of L. sigmodontis invector-derived L3 (vL3), adult males (AM), pre-gravid adult females(PAF), gravid adult females (GAF), and immature Mf (iMf). In addition toidentifying dynamic changes in the ESP profile through the lifecycle, weshow important differences in the adult secretomes of L. sigmodontis andB. malayi, especially in the abundance of two novel proteins released byfemale L. sigmodontis that lack orthologues in B. malayi. As has beenobserved in other parasitic nematodes, we find transthyretin-like family(TTL) proteins to be particularly dominant in the ESP. Leakage ofuterine fluid may account for the remarkable diversity of proteins thatwe detect in GAF ESP, and we highlight several novel proteins thatwarrant evaluation in vaccine trials and as anti-inflammatory mediators.

2.3 Experimental Procedures

Ethical Considerations

All experimental procedures on the animals required for vL3 productionat the Muséum National d'Histoire Naturelle were approved by the ethicalcommittee “Cuvier” (no 68-002) and carried out in strict accordance withEU Directive 2010/63/UE and the relevant national legislation (FrenchDécret no 2013-118, 1 Feb. 2013). All other parasite stages wereharvested from animals maintained at the University of Edinburgh incompliance with a UK Home Office Animals (Scientific Procedures Act)1986 project license and the recommendations of the local ethical reviewcommittee.

Parasites and Protein Preparations

The life cycle of L. sigmodontis was maintained in jirds (Merionesunguiculatus) infected with vL3 harvested from the mite vectorOrnithonyssus bacoti. After 70-90 days, GAF and AM were recovered fromthe pleural cavity by lavage with serum-free RPMI 1640 medium (LifeTechnologies), whereas PAF were recovered 32 days post-challenge. Toharvest iMf liberated in vitro, GAF culture medium was removed after 24h and centrifuged at 1,900 g for 20 minutes (4° C.). Blood-derivedmicrofilariae (bMf) were obtained by overlay of blood (from cardiacpuncture of jirds >75 days post-infection) onto a 25% Percollsuspension, centrifugation at 1,900 g for 20 minutes (4° C.), andpassage of the bMf fraction through a PD-10 desalting column (GEHealthcare) prior to culture. The vL3 larvae were dissected directlyfrom the mite vector and washed three times in RPMI 1640 before transferto culture vessels.

To determine the relative abundance of proteins in the secretome of eachparasite stage, ESP and whole body extracts (WBE) were extracted andanalysed separately. All parasite stages were incubated in serum-freeRPMI 1640 supplemented with 100 U/ml penicillin, 100 μg/ml streptomycinand 1% glucose at 37° C. (5% CO₂) in ultra-low attachment flasks(Corning), and were confirmed to be viable during incubation bymicroscopic examination. The medium was replaced every 24 h, and spentmedia recovered at 24 h and 48 h were centrifuged at 1,900 g for 20minutes (4° C.) in low protein-binding Oak Ridge tubes (polypropylenecopolymer; Thermo Scientific Nalgene) to remove debris. To purifyproteins from the supernatant, hydroxylated silica slurry (StrataCleanResin, Agilent Technologies) was added at 30 μl/ml and vortex-mixed athigh speed for 2 min. Resin used for each 24 h incubation sample wasreused for the respective 48 h sample to concentrate ESP prior tostorage at −80° C. Initial experiments using soluble WBE (used as aproxy for ESP, as limited amounts of the latter were available)displayed no visible differences in protein profiles by SDS-PAGE usingresin-bound protein compared to equivalent unbound material (data notshown). Analyses were performed with separate ESP batches inquadruplicate for GAF, triplicate for AM, and duplicate for PAF, iMf andvL3.

Soluble WBE was prepared by homogenisation in 25 mM ammoniumbicarbonate, 1% RapiGest SF surfactant (Waters) and cOmplete ProteaseInhibitor Cocktail (Roche) using a mini-pestle in a microcentrifugetube. This was followed by 10 cycles of sonication on ice using aVibra-Cell VCX130PB sonicator (Sonics & Materials, Inc.) with microprobe(10 sec sonication alternating with 30 sec incubation on ice).Homogenised samples were centrifuged at 13,000 g for 20 minutes (4° C.)and the supernatant retained. The WBE preparations were obtained fromsingle pools of parasites for all stages except GAF and AM, where twobiological replicates were available. Protein concentrations weredetermined using the Pierce Coomassie Plus (Bradford) Protein Assay(Thermo Scientific).

Surface Biotinylation of Live Worms

Samples of 10 adult male and five female nematodes were washed threetimes with pre-chilled PBS buffer and incubated for 30 min with 1 mMEZ-link Sulfo-NHS-SS-Biotin (Thermo scientific), or PBS only (negativecontrol), at 4° C. with gentle agitation. The biotinylating solution wasremoved, and the reaction quenched with 100 mM glycine in PBS beforewashing the nematodes three times in PBS-glycine. Labelled nematodeswere stored at −80° C. Surface proteins were extracted by sequentialincubations in PBS buffer alone, 1.5% octyl β-D-glucopyranoside (Sigma),0.5% SDS and then 4 M urea (all in PBS) for 1 h each (room temperature).Proteins released at each step were incubated with 30 μl ofhigh-capacity streptavidin-agarose beads (Thermo Scientific) for 2 h atroom temperature with rotary mixing. To recover bound biotinylatedproteins, the supernatant was removed and the beads were washed threetimes in PBS and three times in 25 mM ammonium bicarbonate prior toincubation in 50 mM DTT (Sigma), 25 mM ammonium bicarbonate at 50° C.for 30 min. The supernatant was removed and the DTT diluted tenfoldbefore digestion with 0.2 μg proteomic-grade trypsin (Sigma) overnightat 37° C. The resultant peptides were concentrated using C₁₈reverse-phase spin filters (Thermo Scientific) according to themanufacturer's instructions prior to MS analysis.

To confirm efficient and specific labelling of the parasite surface, AMand GAF were fixed in 70% hot ethanol after subjection to biotin andcontrol labelling as above. Paraffin-embedded sections (4 μm) weredeparaffinised, rehydrated and blocked in 1% BSA and 0.3% Triton X-100in PBS (blocking buffer) for 1 h (room temperature), followed by two5-min washes in PBS with gentle agitation. The sections were incubatedwith streptavidin-FITC (Sigma) at a 1/1,000 dilution in blocking bufferfor 1 hr (room temperature), washed three times, and mounted withProLong Gold anti-fade reagent (Life Technologies). Images were obtainedon an Axio Imager.M2 fluorescence microscope (Zeiss) using Zen 2012software (Zeiss), combining the FITC channel with brightfieldillumination.

Sample Preparation for Proteomics

StrataClean Resin containing bound ESP was washed twice with 25 mMammonium bicarbonate before suspension in 0.1% RapiGest SF, 25 mMammonium bicarbonate. The resin samples were heated at 80° C. for 10min, reduced with 3 mM DTT at 60° C. for 10 min, cooled, then alkylatedwith 9 mM iodoacetamide (Sigma) for 30 min (room temperature) protectedfrom light. All steps were performed with intermittent vortex-mixing.The samples were then digested using 0.2 μg proteomic-grade trypsin at37° C. overnight with rotation, centrifuged at 13,000 g for 5 min, andthe supernatant removed. The resin was washed twice with 0.1% RapiGestSF, 25 mM ammonium bicarbonate and the supernatants pooled. To removeRapiGest SF, the samples were precipitated using TFA (finalconcentration, 1%) at 37° C. for 2 h and centrifuged at 12,000 g for 1hr (4° C.). The peptide supernatant was concentrated using C₁₈reverse-phase spin filters according to the manufacturer's instructions.The WBE samples were reduced and alkylated as above, digested withtrypsin at a protein:trypsin ratio of 50:1 at 37° C. overnight, andprecipitated to remove RapiGest SF as for the ESP preparations.

NanoLC MS ESI MS/MS Analysis

Peptide solutions (2 μl) were analysed by on-line nanoflow LC using thenanoACQUITY-nLC system (Waters) coupled to an LTQ-Orbitrap Velos (ThermoScientific) MS as previously described (13, 26). Thermo RAW files wereimported into Progenesis LC-MS (version 4.1, Nonlinear Dynamics) andspectral data were transformed to MGF files prior to export for peptideidentification using the Mascot (version 2.3.02, Matrix Science) searchengine as detailed previously (26). Tandem MS data were searched againstthe protein predictions from the L. sigmodontis genome and its Wolbachiasymbiont, wLs [obtained from the online nematode genome of L.sigmodontis, release nLs 2.1.2, 10,246 protein sequences (M. Blaxter, S.Kumar, G. Koutsovoulos; unpublished); and release wLs 2.0, 1,042 proteinsequences (27)], together with predicted proteomes for the rodent host(Mus musculus, Uniprot release 2012_08, 16,626 protein sequences; andMeriones unguiculatus, Uniprot release 2012_08, 223 protein sequences)and a general contaminant database (GPMDB, cRAP version 2012.01.01, 115protein sequences). Search parameters, allowable modifications and thefalse discovery rate were defined as reported previously (13, 26).Mascot search results were imported into Progenesis LC-MS as XML filesand analysed according to the following criteria: at least two uniquepeptides were required for reporting protein identifications, and anindividual protein had to be present in ≥2 biological replicates to beincluded in the ESP dataset. Protein abundance was calculated by theiBAQ method; i.e., the sum of all peak intensities from the Progenesisoutput was divided by the number of theoretically observable trypticpeptides (28). For ESP and WBE, protein abundance was normalised bydividing the protein iBAQ value by the summed iBAQ values for thecorresponding sample, and the reported abundance is the mean of thebiological replicates. Normalised peptide intensities rather than iBAQvalues were used to calculate fold-changes between control andbiotinylated worm surface preparations. Mass spectrometric data havebeen deposited in the ProteomeXchange Consortium database(http://proteomecentral.proteomexchange.org) via the PRIDE partnerrepository (29) with the dataset identifier XXXXXXXXX.

In Silico Analyses of Proteins

The domain content of proteins identified in the ESP assessed using Pfam(v. 27.0) with the gathering threshold as a cut-off. A hypergeometrictest for enrichment of Pfam domains in ESP proteins compared with thecomplete predicted proteome of L. sigmodontis was performed using thephyper toolkit within the R programming environment (30). The Benjamini& Hochberg step-up FDR-controlling procedure was applied to thecalculated, adjusted P-values (31). Structural homologues of abundantuncharacterised proteins were identified through comparison to theNational Center for Biotechnology Information non-redundant proteindatabase (DELTA-BLAST search; E-value cut-off 1⁻⁰³) and to the UniProtdatabase (PSI-BLAST search) via the Phyre² protein fold recognitionserver (32). The conserved domain structure of selected, abundant ESPproteins was also interrogated in InterProScan 4 (33). Venn diagramswere created using VENNTURE (34), while for heat-maps, hierarchicalcluster analysis was performed using the (1-r) distance metric inGENE-E. Prediction of classical N-terminal signal peptides,non-classical secretion signatures, mitochondrial targeting sequences,O-glycosylation sites and propeptide cleavage sites was performed usingthe SignalP 4.0 server (35), the SecretomeP 2.0 server (36), MitoProt(37), the NetOGlyc 4.0 server (38) and the ProP 1.0 server (39),respectively. Brugia malayi orthologues of L. sigmodontis proteins weredetermined using reciprocal BLAST with a bit score cut-off of 50.

ShK domains were identified in the complete predicted proteomes of thefilariae B. malayi (9), D. immitis (10), L. sigmodontis, Onchocercaochengi, Acanthocheilonema viteae (draft unpublished genomes availableat http://www.nematodes.org/genomes/; Blaxter et al., unpublished), W.bancrofti and L. loa (11), plus the ascaridid nematode Ascaris suum (40)(which is an outgroup for the filarial species), using the Pfam hiddenMarkov model for the domain and hmmer (version 3.1b.1). Each domain wasexcised and a total of 531 distinct domains identified, which werealigned using ClustalOmega (41). Inspection of the alignment revealedthat a subset of domains were misaligned (and therefore did not have thesix cysteine residues in register with the others); these were correctedmanually. The alignment was analysed for phylogenetic signal usingMrBayes (version 3.2) (42) and two runs of four chains each were run fortwo million generations. The first million generations were discarded asburn-in after inspection in Tracer (version 1.5; A. Rambaut,unpublished: http://tree.bio.ed.ac.uk/software/tracer/) and a consensustree was inferred from the remaining 10,000 samples taken every 100generations. Sequence logos were generated for all 531 ShK domains, alldomains from nLs_04059 and orthologues, and each of the six distinctsets of orthologous domains, using the WebLogo server(http://weblogo.berkeley.edu/) (43).

2.4 Results

Distribution of Proteins in ESP Across Parasite Lifecycle Stages

We searched ˜120,000 MS spectra per lifecycle stage against proteinsequences predicted from the L. sigmodontis and wLs genome assemblies. Atotal of 302 quantifiable filarial proteins (i.e., represented by ≥2unique peptides in ≥2 biological replicates) were detected in ESP acrossthe five lifecycle stages. A majority of these (195 proteins, 64.6%)were uniquely identified in GAF (FIG. 1b ). Hierarchical clustering ofthe proteomic profiles clearly separated ESP and WBE (FIG. 8). The vL3ESP data profile was distinct; not only from that of the other ESPpreparations, but also from vL3 WBE (FIG. 8). The closer clustering ofiMf with PAF ESP rather than GAF ESP was surprising, but may reflect themuch lower complexity of the PAF and iMf ESP datasets. Strikingly,excluding GAF, fewer than six stage-specific proteins each were observedin ESP (Table 1). In vL3, these stage-specific proteins included highlyexpressed vaccine candidates originally identified from L3 of otherfilarial species, including activation-associated secreted protein 1(ASP-1) and abundant larval transcript protein 1 (ALT-1). The functionalidentities of proteins restricted to other lifecycle stages wereunexpected. Thus, PAF released two cuticular proteins and twoantioxidant proteins that were not observed in ESP from GAF (Table 1).The only wLs-derived proteins that were quantifiable in any ESP were twocomponents of the GroELS chaperonin complex, which were found solely inpreparations from PAF and GAF (Table 3).

We explored functional distinctness of ESP from different lifecyclestages by determining protein domain overrepresentation relative to thecomplete predicted proteome of L. sigmodontis. The greatestfold-enrichment scores were observed in the AM ESP, which containedthree proteins with a major sperm protein (MSP) fibre protein 2b (MFP2b)domain and 10 proteins with a TTL family domain (FIG. 2). The TTL familywas also overrepresented in iMf (seven proteins) and PAF (nineproteins). Notably, PAF exhibited significant enrichment forintermediate filament and lamin-tail domains (three members each). TheESP from GAF was enriched for lamin-tail and immunoglobulin I-setdomains, as well as two proteasome and two laminin families (FIG. 2).Overall, iMf, AM and PAF secreted a greater proportion of proteins withrelatively low abundance in WBE than did vL3 and GAF (FIG. 3). However,all of the lifecycle stages exhibited secretomic profiles clearlydistinct from WBE, in that proteins which were highly abundant in ESPtended to be rare in WBE and vice-versa (FIG. 4, FIG. 5). Identificationof proteins in ESP was strongly correlated with sequence featuressuggesting secretion: 31.1% of ESP protein sequences were predicted tobegin with a classical signal peptide, while a further 30.5% werepredicted to contain an internal, non-classical secretion signature.

Abundant Proteins Released by Adult Parasites

The GAF ESP displayed the most complex composition, and the majority ofthe abundant proteins secreted by this stage were uncharacterised orcontained conserved domains associated with very limited functionalinformation (FIG. 4b ). The dominant GAF ESP protein (nLs_03577) wasunique to filarial nematodes and exhibited only very weak similarity toa bacterial P-type ATPase (Tables 4 and 5). Twelve distinct TTL familyproteins were identified in GAF ESP (FIG. 4b ), although only two wereunique to this lifecycle stage. Another abundant GAF ESP protein(nLs_08836), also well-represented in PAF and iMf ESP, contained vonWillebrand factor type-d (VWD) and cysteine-rich (C8) domains in itscarboxy-terminal portion. The best match identified for nLs_08836 was anapolipophorin from A. suum, but nLs_08836 lacks the expectedamino-terminal lipoprotein domain, and the carboxy-terminal portiondisplayed weak similarity to predicted zonadhesin-like or SCO-spondinproteins (Tables 4 and 5). A protein (nLs_04059) that contained sixmetridin-like ShK toxin domains, nLs_04059, was moderately abundant inGAF ESP and was also observed in PAF, AM and iMf secretomes (FIG. 9, 11,12). While the ShK domain has a wide phylogenetic distribution, theparticular pattern apparent in nLs_04059 is limited to filariae (Tables4 and 5; see below for detailed analyses of this protein). Additionalproteins present in GAF ESP were homologues of previously described ESPantigens from other filarial species. However, RAL-2 (44), SXP-1 (45),S3 (46) and CCG-1 (47) remain functionally obscure.

Functionally defined components of the ESP included a small cysteineprotease inhibitor [CPI (48)], the omega-class glutathioneS-transferases [GST (49)], the MSPs (50), and the microfilarial sheathprotein (51) (FIG. 4b ). Additionally, L. sigmodontis homologues of Av33and ES-62, proteins known to be abundant in the ESP from adult femalesof other filarial species, were identified. Av33 is similar to anaspartate protease inhibitor from A. suum (52), whereas ES-62 is asecreted leucyl aminopeptidase (53). A secreted acid phosphatase, whichmay be involved riboflavin metabolism and have a role in the hydrolysisof prosthetic groups such as flavin mononucleotide and/or pyridoxal5-phosphate (54, 55), was prominent in PAF ESP. Three of the GAF ESPproteins had putative lipid-binding regions: ML-domain proteins havebeen reported to interact with cholesterol and lipid A (56, 57), theconserved filarial antigen Ov16 has a putativephosphatidylethanolamine-binding domain (58), and a novel and highlyabundant vitellogenin (nLs_07321) contained an amino-terminal lipidtransport domain.

There was extensive overlap in the identities of the most abundantproteins in the ESP of PAF, AM and iMf compared with that of theparticularly diverse GAF. These less complex ESP mixtures neverthelesscontained dominant components overrepresented in individual stages. InPAF, abundant proteins included several glycolytic enzymes and twoheat-shock proteins, as well as a galectin (β-galactoside-bindingprotein 1) and a highly unusual protein, nLs_03350, containing bothC-type lectin and acetylcholine receptor domains (FIG. 4c ). Abundantcomponents of AM ESP included three isoforms of MFP2 (59) and proteinsknown to be highly expressed in sperm or seminal fluid, such as anextracellular superoxide dismutase (60) and a serine protease inhibitor(61) (FIG. 4a ). However, AM ESP also contained several previouslydescribed but uncharacterised proteins, such as RAL-2 (44), nematodesecreted protein 22U (62) and immunogenic protein 3 (63). A novel KH(RNA-binding) domain protein had homologues in other filarial species,but also weak homology to the Vasa DEAD-box helicase GLH-2 fromCaenorhabditis elegans (Table 4), which is associated with spermatogenicchromatin (64).

Abundant Proteins Released by Larval Parasites

Characterisation of ESP from the bMf stage posed special challenges.Despite the two-stage purification process and prolonged culture invitro, 92.4% (61 of 66) of proteins robustly quantified in bMf ESP werederived from the rodent host. The dominant serum components identifiedwere fibronectin, complement C3, serum albumin, hemopexin, plasminogenand ceruloplasmin; while lower amounts of IgM were also detected (Table6). Of the five quantifiable parasite-derived molecules, three were TTLproteins. To obtain characterise Mf-derived ESP in more depth, weharvested iMf from GAF cultures in vitro, separated them from the femalenematodes, and proceeded with in vitro incubation. This procedureincreased the detection of proteins of nematode origin to 36 (FIG. 5b ),although as expected, the dominant proteins in iMf ESP closely mirroredthe profile of GAF ESP (FIG. 4b ). Interestingly, the two most abundantparasite ESP proteins observed in bMf, a TTL protein and anematode-specific uncharacterised protein (nLs_03443), were not presentin iMf ESP (Table 6). Non-unique but proportionally enriched proteins iniMf included two galectins (β-galactoside-binding proteins 1 and 2), afatty acid and retinoid-binding protein (FAR-1), and a nucleosidediphosphate kinase (FIG. 5b ), all of which are known to be expressedthroughout the filarial lifecycle (65, 66). In addition, Ls110, which issecreted from the uterine epithelium during embryonic development (67),was detected in iMf ESP but not iMf WBE. Conversely, the major sheathproteins Shp1a and Shp4 were found in iMf WBE but were not secreted(supplemental Table S1). Another distinctive feature of the iMf ESP wasthe overrepresentation of two proteoglycan core proteins: aperlecan-like protein that exhibited moderate similarity to UNC-52 fromC. elegans (Table 4 FIG. 5b ) (68); and a chondroitin proteoglycan (CPG)containing six peritrophin-A chitin-binding domains, which was distantlyrelated to C. elegans CPG-2 (69) (Tables 3 and 4). However, a large(˜250 kDa predicted mass) plasminogen-apple-nematode (PAN) domainprotein, which displayed weak similarity (Table 4) to the predictedmucin SRAP-1 from C. elegans (70), was more abundant than either of theproteoglycans in iMf ESP (FIG. 5b ). Finally, an apparently novelperoxidasin-like protein with orthologues in other filarial nematodesand more distant relatives in A. suum and Caenorhabditis briggsae (Table4) was also identified in iMf ESP (FIG. 5b ).

Although ESP from the vL3 stage was the least diverse dataset in ourstudy, it showed a distinctive repertoire of highly abundant proteins.Thus, vL3 ESP was composed of previously characterised filarial proteinsthat are known to be uniquely expressed or enriched in this stage [suchas ASP-1 (71), ALT-1 (72), and cathepsin-L-like protease (73)], andother antigens that were well represented in ESP from other stages(RAL-2, CPI-2, Ov16 and β-galactoside-binding proteins) (FIG. 5a ). Thenematode secreted protein 22U was moderately abundant in the L.sigmodontis vL3 ESP preparations (FIG. 5a ), but apparently is notexpressed in vL3 of other filarial species (62). This stage may berelatively quiescent in terms of secretory activity until they adapt tothe mammalian host and undergo the third moult. Analysis of ESP frommoulting L3 identified fivefold more proteins than from vL3 ESP in B.malayi (17).

Phylogenetics of Novel, Filaria-Specific ESP Proteins

The most abundant protein in GAF ESP, nLs_03577, is an enigmatic,uncharacterised molecule with a predicted MW of 28.5 kDa and a lack ofconserved domains, with the exception of a classical N-terminal signalpeptide. Downstream of the signal peptide, moderate to high levels ofsequence conservation were apparent across the Filarioidea in theN-terminal portion (FIG. 10). However, the C-terminal segment displayedlow complexity and was highly variable between filariae, with twoisoforms in B. malayi diverging in this region only (FIG. 10). The L.sigmodontis protein was predicted to contain six potential N-linked and11 O-linked glycosylation sites, as well as propeptide cleavage sites atpositions 31 and 147. The former cleavage site was absolutely conservedwithin the Filarioidea, despite some variation in the motif, whereas thelatter (at position 154 of the consensus) was unique to L. sigmodontis.These observations suggest that several processed isoforms of nLs_03577might be secreted by L. sigmodontis. Phylogenetic analysis of nLs_03577orthologues confirmed that this protein is restricted to theFilarioidea, with no representatives in A. suum or other non-filarialnematodes. The base of the tree was poorly resolved due to the lack ofsignal in the C-terminal portion (FIG. 11). However, nLs_03577 clearlyclustered with an orthologue in the rodent filaria, A. viteae, whileorthologues in D. immitis and Onchocerca spp. formed the most distantgrouping (FIG. 11).

The ShK domain protein nLs_04059 was a particularly distinctive moleculeidentified in all ESP preparations except vL3. One other L. sigmodontisShK domain protein, the astacin protease nLs_03368, was a rare componentof GAF ESP only (Table 3). The ShK domain (or metridin-like toxindomain, also known as the SXC or six-cysteine domain) was firstidentified in cnidarian venoms, but is particularly abundant in nematodeproteomes (74), where it is associated with secreted proteins. Theprototypic ShK peptide (from the cnidarian Stichodactyla helianthus) isa type 1 toxin that blocks voltage-gated potassium channels, andsynthetic analogues are currently under development as a therapy forautoimmune diseases, in which Kv1.3 channels expressed by effectormemory T-lymphocytes are specifically targeted (75). Although nLs_04059was not especially abundant in any ESP preparation, its presence in thesecretomes of all mammalian-derived stages and its unusual domainstructure (FIG. 9) suggest a potentially immunomodulatory role.

The nLs_04059 protein has the largest number of ShK domains (six) of anyprotein in L. sigmodontis. We identified orthologous genes in all theother filarial nematode genomes, each containing six ShK domains (FIG.12). The nLs_04059-like ShK domains form a distinct subset of allfilarial and A. suum ShK domains (I FIGS. 12 and 13), with a strikingpattern of conservation particularly around the last threeuniversally-conserved cysteine residues (FIG. 6). A proline residue (atposition 32 of the alignment, but residue 17 of the nLs_04095 domains)was also strikingly conserved in the nLs_04059 domains (FIG. 6), but notcommon in the full set of 531 domains. In nLs_04059 and its orthologues,the six ShK repeats are separated by five low-complexity spacers (27-104amino acids) (FIG. 9). Some spacer domains were conserved, but othersshowed variation in the pattern and length of low complexity,serine-rich regions. The spacer domains have no clear similarity toother proteins, but by analogy to the ShK mucins of the ascarididToxocara canis (76), they could be recipients of O-linked glycandecorations. There are 65 potential O-glycosylation sites on nLs_04059.However, by geLC-MS we found that nLs_04059 migrated exclusively at theexpected molecular weight of the unmodified mature protein (˜52 kDa),ruling out a mucin-like structure (data not shown). This protein alsocontained two lysyltyrosine dyads located within the C-terminal ShKdomain (FIG. 9). Since a lysyltyrosine dyad is essential for binding oftype-1 cnidarian peptide toxins to potassium channels (77), this couldbe related to Kv1 channel-blocking activity. Notably, one lysyltyrosinedyad in ShK domain 6 is conserved in many (although not all) orthologuesin other species (FIG. 6).

Proteins Associated with the Adult Nematode Surface

The nematode cuticle is the critical interface between the parasite andthe immune system of its host (78). Surface-associated proteins maysimply mirror ESP, perhaps by passive adsorption of released material,or comprise a distinct component of the exoproteome. Live AM and GAFnematodes were surface-labelled incubated with Sulfo-NHS-SS-Biotin andfractionated. Immunofluorescent imaging of fixed nematode sectionsconfirmed that biotin labelling was largely confined to the cuticularlayers (FIG. 14). Low levels of endogenous biotin were present withininternal structures as expected. We identified five proteins that werepresent in biotin-labelled AM extracts but not unlabelled controls and11 proteins in biotin-labelled GAF (Table 2). In addition, a furtherfour (AM) and 39 (GAF) proteins were enriched by more than 50-fold inbiotin-labelled samples relative to unlabelled controls (Table 7),suggesting that these molecules were abundant on the parasites' surfacebut may also be associated with endogenous biotin. There wasconsiderable overlap between ESP and biotin-labelled protein profiles inboth sexes. However, AM and GAF displayed two and 12 proteins,respectively, that were uniquely present in surface-labelled extracts(Table 2 Table 7). Conversely, many of the highly abundant ESP proteins,such as nLs_03577, the vitellogenin nLs_07321, uncharacterised proteinS3 and ES-62 were not detected in biotin-labelled extracts.

A striking feature of the surface-associated proteins was the presenceof two ectoenzymes involved in purinergic signalling. These were anadenylate kinase predominant in AM extracts and a purine nucleosidephosphorylase found exclusively found in GAF extracts (79) (Table 2,Table 7). A homologue of complement component 1, q subcomponent-bindingprotein was identified in GAF surface-labelled extracts. Like the humanhomologue, the L. sigmodontis protein contained an N-terminalmitochondrial import signal sequence, although the former is expressedin a number of extramitochondrial locations, including on the surface oflymphocytes, endothelial cells, dendritic cells and platelets (80).These proteins may play a role in immunomodulation, as purinergicsignalling is known to regulate lymphocyte trafficking (79), while thecomplement component 1q receptor is involved in vasodilation via thegeneration of bradykinin (80).

Surface extracts from AM contained a homologue of the actin-bindingprotein, calponin, which has been localised to both striated muscle andthe cuticle in adult O. volvulus (81). The GAF surface extractscontained two proteins, protein disulphide isomerase and a leucine-richrepeat family protein, both of which have previously been associatedwith cuticle synthesis in filariae and C. elegans (82, 83). Stressresponse-related proteins were also well represented on GAF (includingthioredoxin peroxidase (84), aldehyde dehydrogenase, a thioredoxin-likeprotein and heat-shock proteins), as were several enzymes of pyruvatemetabolism (Table 2 and Table 7). Notably, the endosymbiont-derivedWolbachia surface protein was found to be accessible to surfacebiotinylation in GAF.

Comparison with the Secretome of Adult B. malayi

The ESP from several lifecycle stages of B. malayi have been describedpreviously (14, 16, 17). In these three studies, the only common stagewas adult [with both sexes cultured together in (14)]. Of 297 proteinsidentified in adult L. sigmodontis ESP, 92.6% had an orthologue in B.malayi. However, the majority (61.6%) of these B. malayi orthologueswere not observed in the B. malayi secretome (FIG. 7a ). Analysis ofPfam domains failed to indicate any significant enrichment in thisunique dataset (data not shown). Conversely, although each B. malayistudy revealed a surprising number of study-specific secreted proteins,orthologues of the proteins reported in all three B. malayi secretomeswere also detected in adult L. sigmodontis ESP (FIG. 7a ). This commoncore included leucyl aminopeptidase, enolase, triosephosphate isomerase,β-galactoside-binding protein 1, acetylcholine receptor protein,cyclophilin-5 and macrophage migration inhibitory factor-1. The 22 L.sigmodontis adult ESP proteins that lacked B. malayi orthologues (FIG.7b ) included two of the most highly abundant GAF ESP molecules (thevitellogenin nLs_07321 and the VWD protein nLs_08836), together withsecretory protein Ls110 and two superoxide dismutase isoforms. Althoughthe B. malayi secretome studies identified a total of 90 proteins thatdid not have orthologues in L. sigmodontis (FIG. 7b ), only one(cuticular glutathione peroxidase) was observed in all three studies.Since standardised quantification methods were not used for our L.sigmodontis and the published B. malayi studies, it is difficult todetermine whether adult B. malayi and L. sigmodontis differ in theirlevels of secretion for individual ESP proteins. However, in terms ofrank abundance, triosephosphate isomerase, macrophage migrationinhibitory factor-1, and γ-glutamyl transpeptidase were reported to begrossly overrepresented in adult B. malayi; whereas adult L. sigmodontisESP was enriched for uncharacterised protein nLs_03577 (orthologous toBm1_38495), TTL protein nLs_09750 (orthologous to Bm1_43635), andhomologues of Av33 and S3 (Table 3). Proteins that were apparentlyequally abundant in relative terms between each species included leucylaminopeptidase and homologues of CPI-2 and Ov16.

2.5 Discussion

Quantifying the Secretomes of a Model Filarial Nematode

Filarial nematodes exact a significant burden of morbidity in humanpopulations and are important pathogens of companion animals. Whileefficacious anti-filarial drugs exist, the spectre of the evolution ofgenetic resistance to these is ever-present (5, 6), and alternativeroutes to treatment are required. It would be preferable to be able toprevent infection as well as treat patent disease, and thus ananti-filarial vaccine would be an extremely valuable addition to medicaland veterinary treatment options (85). The ESP released by parasitesinto their hosts have been the target of vaccine development fordecades, but the understanding of these molecules in filarial nematodesis limited. Whereas previous studies have catalogued the proteinsinferred to be present in filarial ESP, quantitative assessments oftheir abundance have not been explored previously using anintensity-based approach. Using the model rodent filarial nematode L.sigmodontis, it is possible to prepare material from across the nematodelifecycle, and thus examine the different vertebrate-parasitic stages indetail. Applying semi-quantitative MS analysis of ESP, we identifiedsecreted proteins and determined their abundance, limiting our analysisto 302 proteins that could be robustly quantified using ≥2 uniquepeptides.

The Secretome of Adult Nematodes

In L. sigmodontis, GAF was responsible for the majority of ESP proteomicdiversity. The other four lifecycle stages examined contributed only 11proteins (3.6% of the total) that were not present in GAF ESP. Thisfinding contrasts with a qualitative analysis of B. malayi secretomescomparing adults, Mf and L3, and incorporating data obtained fromsingle-peptide hits, which found that Mf contributed the greatestproportion of unique proteins (17). However, an earlier assessment ofthe B. malayi GAF, AM and Mf secretomes concluded that GAF produced thegreatest number of unique hits (16), suggesting that methodologicaldifferences may underlie these contrasting results. The diversity of GAFESP is consistent with the material containing not only somatic adultESP, but also proteins released from the reproductive tract that derivefrom the processes of oogenesis, fertilisation and embryonic developmentin utero (all filarial pathogens are ovoviviparous).

Nematode sperm are acutely sensitive to aerobic damage (86). The AM ESPcontained proteins suggestive of roles in protection of sperm againstoxidants and other stressors, including superoxide dismutase, a serineprotease inhibitor and a glutaredoxin-like protein. Glutaredoxins arethiol-containing antioxidant proteins, and C. elegans GLRX-21 plays akey role in mitigating selenium toxicity (87). Mammalian seminal fluidaccumulates selenium, which if in excess, can impede sperm motility(88). A homologue of the serine protease inhibitor is secreted by A.suum during the acquisition of motility and contributes to spermcompetition by inhibiting the activation of surrounding spermatids (89).Lysis of sperm during aerobic culture may account for the high levels ofMSPs observed in AM ESP and in ESP obtained from PAF, GAF and iMf.Female nematodes are fertilised some weeks before the first Mf areproduced (90), and the dominance of MSPs in PAF ESP indicates thatleakage of sperm from the female reproductive tract occurs beforeparturition.

Several unique antioxidant proteins (nucleoredoxin-like protein-2,glutathione reductase and translationally-controlled tumour protein)were found in PAF ESP, suggesting an enhanced requirement for protectionduring this stage. In B. malayi, homologues of the nucleoredoxin-likeproteins, which resemble large thioredoxins (91), are present in ESP butdo not exhibit stage-specific expression (92). Two unique cuticlebiosynthesis related proteins were also released by PAF, suggesting thatcuticular remodelling occurs during their final stages of growth. Thismay result in increased susceptibility to immune-driven oxidative stressor damage during copulation (93). Heat-shock proteins, which wereoverrepresented in PAF ESP, were detected previously in B. malayi adultnematode ESP (94).

The Mature Microfilarial Secretome is Dominated by Host Proteins

In many filarial nematodes, microfilariae are enclosed in aproteinaceous sheath comprising an inner layer that originates from theeggshell and an outer layer that is produced by secretions in the distalportion of the uterus. Five major structural proteins have beenidentified in the L. sigmodontis sheath, some of which are synthesisedin the developing embryo and others in the uterine epithelium (51), butnone of these were found in iMf ESP, indicating that they are stablecomponents. Many host serum proteins were released from bMf in culture.These are likely to derive from specific interactions with the parasitesurface, perhaps reflecting a tension between the nematode exploitingthe host and the host immune system recognising the parasite. Thefinding of host at the Mf surface is not new, as five serum componentswere only proteins released by SDS extraction of L. sigmodontis Mfsheaths (95), and human serum albumin has been detected on the sheathsurface of W. bancrofti Mf (96), but is generally not found on Brugiaspp. Mf (97). The L. sigmodontis sheath is permeable to molecules of upto 70 kDa (98), and therefore might retain some host proteins aftertransfer to culture. However, several abundant serum proteins that wedetected in bMf ESP are considerably larger than this (for example,ceruloplasmin and fibronectin) and thus must be either adsorbed onto thesheath surface or proteolytically processed prior to uptake. Hemopexinand ceruloplasmin have roles in heme and copper transport (99),respectively; hence, they may be exploited as a source of essentialcofactors by the parasite.

Several parasite-derived products were identified as secreted by iMf,including Ls110 [a protein localised in the uterine lumen and variablypresent on iMf, but absent from bMf (67)] and two possible proteoglycancore proteins. Accordingly, large glycoproteins (˜200 kDa) have beendescribed from B. malayi ESP (100). The closest C. elegans homologue ofthe perlecan-like proteoglycan, UNC-52, is a major component of thebasement membrane of contractile tissues, including the pharynx and anusin developing embryos and subsequent stages (68). The L. sigmodontisiMf-derived CPG-like protein is predicted to have chitin-binding domainsand may function in eggshell and sheath development. In C. elegans, CPGsform an inner layer that binds to the central chitinous layer of theeggshell, maintaining the perivitelline space around the embryo (101)forming a barrier to prevent polyspermy (102). In L. sigmodontis, chitinhas been detected in the oocytes and zygotes, although it is absent fromthe iMf sheath (103). The degradation of chitin during Mf sheathdevelopment in utero may release the underlying CPG, which is highlysoluble (101), into the surrounding milieu. The origin and roles two ofthe other novel proteins that were enriched in iMf ESP is less clear.The closest homologue in C. elegans of the PAN domain protein is SRAP-1,which is expressed in the hypodermis, central nervous system and vulvaof developing larvae and is secreted onto the cuticle surface duringmoulting (104). In C. elegans, peroxidasin PXN-2 is located in theextracellular matrix and is required for late embryonic elongation,muscle attachment, and motoneuron axon guidance choice (105).

The Abundant Uncharacterised Proteins Released by Gravid Adult FemaleNematodes

We identified four abundantly secreted or excreted proteins, foundpredominantly in GAF and iMf ESP, that had not been reported previously.Two have only marginal similarity to other proteins: nLs_03577, whichdisplayed a significant match to a P-type ATPase (but lacked an ATPasedomain), and nLs_08836, which showed some similarity to zonadhesin, aVWD protein located in the head of mammalian sperm (106). However, wenote that nLs_08836 is not an orthologue of the C. eleganszonadhesin-domain protein, DEX-1 (107). The third novel protein,nLs_07321, is a vitellogenin. In C. elegans, vitellogenins are expressedexclusively in the intestine, where they bind cholesterol and transportit via the body cavity to the gonad (108). Subsequently, oocytesinternalise the protein and its lipid cargo by receptor-mediatedendocytosis and store it in yolk granules (108). Several vitellogeninshave also been identified in ESP derived from adults of the oviparousgastrointestinal nematode, Heligmosomoides polygyrus (109). The fourthprotein, the ShK domain protein nLs_04059, was distinct from otherproteins containing this motif in nematodes, both in the number ofdomains and their specific sequence. Its relative abundance,distinctiveness and presence in all the filarial species surveyedsuggest that it may be a viable vaccine candidate for both humanfilarial diseases and canine heartworm. Its role in vivo may be tointerfere with the development of acquired immunity by inhibiting theKv1 channels of memory T-cells in a manner analogous to the activity ofcnidarian ShK toxins (75).

The enigmatic TTL family has emerged as one of the most typical andwidespread findings in ESP from both zoo- and phytoparasitic nematodes(110). In C. elegans, there are 63 transthyretin genes, many of whichare secreted and apparently upregulated in response to infectiouschallenge, but only TTR-52 has been ascribed a physiological function[phagocytosis of apoptotic cells (111)]. In the phytoparasite Radopholussimilis, Rs-ttl-2, which is closely similar to one of the most abundantL. sigmodontis TTL proteins (nLs_07576; found in ESP from all stagesexcept vL3 in our study), was localised to the ventral nerve cord (112).A second R. similis TTL family member, Rs-ttl-1, was expressed only inthe vulval region (112), and a homologue of this molecule (nLs_07332)was detected in iMf WBE only. Furthermore, in the ruminant parasiteOstertagia ostertagi, a TTL family (Oo-TTL-1) was a major component ofESP and could be immunolocalised to the pseudocoelomic fluid of adultworms (113). In our study, a L. sigmodontis homologue of Oo-TTL-1(nLs_09750) was abundant in all ESP preparations except those of vL3.

Uterine Fluid as a Source of Nematode and Endosymbiont Products

Proteins excreted or secreted from filarial nematodes could be derivedfrom a number of routes. In addition to oral secretions from theoesophageal glands and release of faecal material from the anus,nematodes also secrete material from the anterior sensory glands(amphids) (114) and the secretory pore, and may also void material fromthe genital openings during copulation and release of Mf. Proteins canalso be released from the hypodermis through transcuticular secretion(115), especially during moulting, and exosome release may also beimportant (116). From our data, we suggest that vulval excretion is themain source of ESP proteins in GAF and PAF, and that the iMf are coatedwith proteins secreted by the uterine epithelium. This interpretation issupported not only by the abundance of MSPs and vitellogenin in GAF andPAF ESP, but by the presence of omega-class secreted GST isoformsexclusively in GAF ESP, which in O. volvulus are only produced byembryos at the morula stage (49). Similarly, ESP proteins in the malenematode probably originate primarily from seminal fluid. Immune serafrom rodents infected with A. viteae react most strongly with male andfemale gonad tissues, including the fluid channels between developingembryos and on sperm in both the spermatheca and seminal vesicle (117).

The role of the Wolbachia endosymbionts of filariae remains unclear: arethey nutritional commensals, supporting the nematode through provisionof energy or cofactors, or part of the immunological avoidancemechanisms of the parasite, or both (13)? It has been proposed thatWolbachia may be present in uterine fluid (118), inside degeneratingembryos (119), or exit via the secretory pore (120). Additionally, theymay secrete proteins into structures that lack bacterial cells, such asthe cuticle (121). Wolbachia-derived proteins were present in very lowamounts in B. malayi secreted products (17). We identified WolbachiaGroELS components in ESP of PAF and GAF, but not in other lifecyclestages. GroEL is the most abundant protein in Wolbachia (13, 15), andits detection in ESP may be through release of whole bacterial cells,for example in the female uterus from degenerating oocytes or embryos,or through secretion. GroEL, as a chaperonin, would be expected to beconfined to the cytosol, although GroEL homologues have been reported to“moonlight” on the surface of some bacterial species (122). We alsodetected Wolbachia surface protein by surface labelling of adult L.sigmodontis, as has been reported in B. malayi (121). This protein is aputative ligand of Toll-like receptors 2 and 4 (119), and these findingssupport the hypothesis that Wolbachia modifies and perhaps misdirectsthe immune response to filariae (123). Whether Wolbachia GroEL alsostimulates proinflammatory Toll-like receptors has not been evaluated,but a precedent exists in other bacteria (124), and antibodies againstthis protein are associated with pathology in LF (125).

The L. sigmodontis Secretome and Vaccine Development for Filariases

For several decades, vaccine development for human and veterinaryfilariases has focused on the L3 stage because irradiated L3 are highlyefficacious at inducing protective immunity (23, 126) and strong anti-L3immunity may block parasite establishment. Litomosoides is an excellentmodel for L3 vaccine research, as the L3 expresses a very similarrepertoire of genes to the human and veterinary pathogens (127).Analyses of ESP from L3 of L. sigmodontis aid in defining astereotypical secretomic profile for this stage. However, no definedparasite antigens (whether alone or in combination) have reproduciblyattained an equivalent level of protection to irradiated L3 in anyfilarial system (7). Furthermore, since a single pair of adult nematodescan generate a patent infection, vaccines directed solely against L3face a potentially insurmountable challenge.

Targeting of Mf has the potential to block transmission, and in the caseof onchocerciasis, to reduce disease pathology. Moreover, the Mf stagehas been shown to be more vulnerable to protective immune responses thanL3 in several vaccination trials (128-130). Vaccination with acombination of ALT-1 and CPI-2 delivered as a DNA vaccine reducedcirculating Mf levels by up to 90% in L. sigmodontis. Importantly, thisprotection was only achieved if immunomodulatory domains of the antigenswere ablated (by mutation or deletion of the coding sequence) and wasmaintained even when the adult nematode burden was not significantlyreduced. This phenomenon was probably to be due to the immunomodulatoryeffects of the native (active) proteins, as transplantation of a singleadult female worm is sufficient to prevent clearance of injected Mf innaïve hosts (131). We suggest that it is likely that many of the otherabundant molecules secreted by GAF may similarly have roles infacilitating Mf survival and could be targeted in an “anti-fecundity”vaccination strategy. Furthermore, the proteins identified by surfacelabelling of the GAF cuticle may also participate in generating apermissive environment (79, 80); thus, vaccination against thesemolecules, if sufficiently divergent from host homologues, might impedeparasite establishment.

2.6 Conclusions

We have shown that L. sigmodontis, especially the GAF stage, releases aremarkable diversity of proteins into the external milieu and themajority of these molecules are uncharacterised. Although many of theseproteins may be involved in fundamental aspects of embryogenesis, asubset are likely to be active immunomodulatory agents that protect thenematodes (and especially the circulating Mf) from the host immuneresponse. The abundant ESP protein, CPI, may represent an archetype forthis dual functionality, as it plays fundamental roles in oogenesis andfertilisation not only in parasitic nematodes but also in C. elegans(132). This suggests that its immunomodulatory properties are an exampleof secondary adaptation to a radically different environment. Thus, thepharmacopeia released by GAF may provide the ideal set of molecule(s) totarget for immunoprophylaxis and chemotherapy of filariases; moreover,it could provide new compounds to tackle proinflammatory and autoimmunediseases (22)

TABLE 1 Proteins unique to the excretory-secretory products ofindividual lifecycle stages of Litomosoides sigmodontis Parasite stageESp^(a) Locus tag Annotation PAF nLs_02441 Epicuticlin nLs_07093Nucleoredoxin-like protein-2 nLs_03968 Nematode cuticle collagenN-terminal domain containing protein nLs_06052 Translationallycontrolled tumor protein nLs_00526 Glutathione reductase AM nLs_07249Glutaredoxin-like protein vL3 nLs_06400 Activation-associated secretedprotein-1 nLs_09374 Abundant larval transcript-1 protein nLs_03087Cathepsin L-like precursor nLs_06524 Calmodulin iMf nLs_02254 MSPdomain-containing protein ^(a)Data for excretory-secretory productsunique to gravid adult females are not shown due to the large number ofproteins (195) in this category (see Table 3). ESP, excretory-secretoryproducts; PAF, pre-gravid adult female; AM, adult male; vL3,vector-derived third-stage larvae; iMf, immature microfilariae.

TABLE 2 Putative surface-associated proteins detected in biotin-labelledadult worm whole body extracts that were absent from unlabelled controlsPeptides Parasite used for Confidence Presence stage Treatmentquantitation score Locus tag Annotation in ESP AM OG 7 857.06 nLs_06907Adenylate No kinase isoenzyme 1 OG 4 417.27 nLs_09715 Major sperm Yesprotein OG 2 186.68 nLs_01742 Filarial antigen No Av33 OG 2 297.13nLs_08458 Filarial antigen Yes Ov16 SDS 2 308.12 nLs_07359 Calponinactin- No binding domain containing protein GAF OG 2 233.80 nLs_09095Protein No disulphide isomerase SDS 2 86.22 nLs_08755 Leucine-rich Norepeat family protein SDS 3 118.56 nLs_09715 Major sperm Yes protein SDS2 99.43 nLs_02353 Complement No component 1, q subcomponent- binding,mitochondrial- like SDS 2 61.97 nLs_01344 Thioredoxin No peroxidase 1SDS 2 108.87 nLs_07321 Vitellogenin Yes PBS 2 309.24 nLs_00851 DNArepair Yes protein Rad4- containing protein PBS 2 309.24 nLs_07061 Heatshock 70 Yes kDa protein PBS 2 309.24 nLs_09360 FMN-binding No domainprotein PBS 3 463.79 nLs_01364 Transthyretin- Yes like protein, partialPBS 2 309.24 nLs_03263 Thioredoxin Yes domain- containing protein ESP,excretory-secretory products; AM, adult male; OG, octylβ-D-glucopyranoside; GAF, gravid adult female; FMN, flavinmononucleotide.

TABLE 3 Protein predictions from the WLS genome Normalised iBAQ valueswLs acc Description GAF ESP GAF WBE PAF ES PAF WBE AM ESP wLs_340co-chaperonin GroES 0.001135899 0.006317778 0.004180529 0.008366027wLs_2830 molecular chaperone GroEL 0.000349505 0.001423165 0.0002793780.011718142 wLs_3910 Outer surface protein Wsp 0.001218499 0.005119482wLs_4630 hypothetical protein 0.000583035 0.001471999 wLs_5240hypothetical protein Wbm0603 0.000387749 0.001641741 wLs_1920 50Sribosomal protein L7/L12 0.000380102 wLs_9520 thloredoxin 0.000169733wLs_930 Outer membrane protein, pal-like 0.000133493 0.000208532wLs_4010 molecular chaperone OnaK 4.7978E−05 0.000214183 wLs_1320hypothetical protein Wmb0010 3.3069E−05 0.000286269 wLs_5680 isoprenoidbiosynthesis protein 9.91181E−06  0.000233577 with amidotransferase-likedomain wLs_9680 elongation factor Tu 0.000638185 wLs_8450 hypetheticalprotein Wbm0655 0.000190391 wLs_5270 superoxide dismutase, SodA0.000182807 wLs_1650 nucloeoid DNA-binding protein 9.97899E−05 wLs_5000heat shock protein 90 1.99158E−05 Normalised iBAQ values wLs acc AM WBEvL3 ESP vL3 WBE IMf ESP IMF WBE wLs_340 0.000987231 0.0048985280.007004044 wLs_2830 0.000305902 0.001431935 0.005708963 wLs_39100.000169721 0.003337764 0.002241786 wLs_4630 0.000104917 wLs_52402.49886E−05 0.00097545 0.004069358 wLs_1920 wLs_9520 wLs_930 3.65115E−050.000291644 wLs_4010 0.000236783 wLs_1820 1.90627E−06 1.60884E−050.000626075 wLs_5680 wLs_9680 wLs_8450 wLs_5270 wLs_1650 0.00025721wLs_5000

TABLE 4 Homologues of abundant Litomosoides sigmodontisexcretory-secretory proteins identified by DELTA-BLAST (National Centrefor Biotechnology Information) Max. Identity Query cover QueryFilter^(a) Top annotated hit^(b) [species] and accession score (%) (%)E-value nLs_00113 AT PAN domain containing protein [Brugia malayi]XP_001900239.1 652 37 77 0.0  FE Flagellin [Salmonella enterica]WP_023208887.1 134 15 12 2⁻²⁸ CO Protein SRAP-1, isoform a [C. elegans]NP_496398.3 114 26 56 3⁻²⁴ nLs_01398 AT Protein UNC-52, Isoform m[Caenorhabditis elegans] NP_001254444.1 1848 52 97 0.0  nLs_02001 AT KHdomain-containing protein [Loa loa] EFO27012.2 513 75 58  2⁻¹⁷⁴ FE Farupstream element-binding protein 1-like [Setaria italica] XP_004972470.197.4 18 65 5⁻¹⁸ CO RNA helicase GLH-2 [C. elegans] AAB03337.1 72.0 25 332⁻¹² nLs_03577 AT Hypothetical protein Bm1_38495 [Brugia malayi]XP_001899152.1 128 60 100 2⁻³² FE Heavy metal translocating P-typeATPase [Dorea sp. 5-2] WP_016217557.1 63.5 29 74 2⁻⁰⁸ CO Protein THOC-2[C. elegans] NP_498392.2 42.0 27 55 1⁻⁰³ nLs_04059 AT Hypotheticalprotein LOAG_17826 [Loa loa] EJD74931.1 262 51 87 7⁻⁸⁰ FE A disintegrinand metalloproteinase with thrombospondin motifs 3-like [Aplysia 52.0 2968 7⁻⁰⁴ californica] XP_005091919.1 CO —^(c) — — — — nLs_05850 ATHypothetical protein LOAG_04060 [Loa loa] XP_003139645.1 269 54 94 1⁻⁸⁰FE Chondroitin proteoglycan 2 [Ascaris suum] EBRG86992.1 247 25 98 1⁻⁶⁸CO CBR-CPG-2 protein [C. briggsae] XP_002633936.1 218 20 93 8⁻⁶⁵nLs_08836 AT Apolipophorin [Ascaris suum] ERG86007.1 1535 42 99 0.0  FEZonadhesin-like [Saccoglossus kowolevskii] XP_002738323.1 256 19 44 4⁻⁶⁵CO Protein VIT-4 [C. elegans] NP_508612.1 97.1 21 8 9⁻²⁰ nLs_01626 ATAnimal heme peroxidase [Loa loa] XP_003141164.1 1367 84 98 0.0  FEPeroxidasin-like protein [Ascaris suum] ERG87495.1 1308 72 98 0.0  COCBR-PXN-2 protein [C. briggsae] XP_002644069.1 1093 47 99 0.0  AT, alltaxa; FE, Filarioidea excluded; CO, Caenorhabditis only. ^(a)Filterswere applied only where the top hit was to taxa othe than Caenorhabditisspp. ^(b)Only annotated hits are shown for non-filarial proteins.^(c)The only hits were to hypothetical proteins containing ShK domains.

TABLE 5 Homologues of obondont Litomosoides sigmodontisexcretory-secretory proteins identified by PSt-BLAST (Phyre²) UniRef50Normalised identity Query Tap annotated hit [species] ID Bits (%)E-value nLs_02001 Transcription elongation factor SPT5 POCR70 135 14.93⁻³⁰ [Cryptococcus neoformans var. neoformans serotype D] nLs_04059Sortilin-related receptor [Homo sapiens] Q92673 210 10.0 1⁻⁵² nLs_08836SCO-spondin [Dania rerio] B3LF39 351 10.4 1⁻⁹⁴

TABLE 6 Quantifiable proteins present in the excretory-secretoryproducts of blood-derived microfilarie Peptides used for ConfidenceNormalised Accession|Gene name quantification score Description(species) iBAQ Q91X72|HEMO_MOUSE 7 898.19 Hemopexin (Mus musculus)1.44⁻⁰² Q8VCM7|FIBG_MOUSE 8 1040.62 Fibrinogen γ chain (Mus musculus)1.15⁻⁰¹ Q8KDE8|FIBB_MOUSE 9 1594.25 Fibrinogen β chain (Mus musculus)9.56⁻⁰² O35090|ALBU_MERUN 29 5013.66 Serum albumin (Merionesunguiculatus) 6.09⁻⁰² P70274|SEPP1_MOUSE 3 203.64 Selenoprotein P (Musmusculus) 5.76⁻⁰² Q61147|CERU_MOUSE 12 2215.19 Ceruloplasmin (Musmusculus) 5.56⁻⁰² P29788|VTNC_MOUSE 6 1035.62 Vitronectin (Mus musculus)5.43⁻⁰² Q61702|ITIH1_MOUSE 7 1236.34 Inter-α-trypsin inhibitor heavychain H1 (Mus musculus) 5.32⁻⁰² P11276|FINC_MOUSE 49 8184.33 Fibronectin(Mus musculus) 3.80⁻⁰² P01027|CO3_MOUSE 28 3368.39 Complement C3 (Musmusculus) 3.63⁻⁰² P01942|HBA_MOUSE 2 149.76 Hemoglobin subunit α (Musmusculus) 3.60⁻⁰² P97515|PETUA_MERUN 6 665.57 α-2-HS-glycoprotein(Meriones unguiculatus) 3.24⁻⁰² P20918|PLMN_MOUSE 13 2062.8 Plasminogen(Mus musculus) 3.13⁻⁰² P13020|GELS_MOUSE 5 1587.22 Gelsolin (Musmusculus) 2.83⁻⁰² Q62577|AMBP_MERUN 6 1120.89 Protein AMBP (Merionesunguiculatus) 2.33⁻⁰² P01029|CO4B_MOUSE 11 1814.14 Complement C4-B (Musmusculus) 1.52⁻⁰² P05367|SAA2_MOUSE 4 820.91 Serum amyloid A-2 protein(Mus musculus) 1.34⁻⁰² Q61703|ITIH2_MOUSE 7 1223.42 Inter-α-trypsininhibitor heavy chain H2 (Mus musculus) 9.20⁻⁰³ nLs.2.1.2.t10069-RA 4264.21 Transthyretin-like protein, partial (Litomosoides sigmodontis)9.11⁻⁰³ P04186|CFAB_MOUSE 6 361.74 Complement factor B (Mus musculus)8.08⁻⁰³ P52430|PON1_MOUSE 2 166.19 Serum paraoxonase/arylesterase 1 (Musmusculus) 7.36⁻⁰³ Q02105|C1QC_MOUSE 2 292.88 Complement C1q subcomponentsubunit C (Mus musculus) 6.31⁻⁰³ P06909|CFAH_MOUSE 2 156.16 Complementfactor H (Mus musculus) 5.81⁻⁰⁶ P05017|IGF1_MOUSE 2 458.52 Insulin-likegrowth factor I (Mus musculus) 5.69⁻⁰⁶ P14106|C1QB_MOUSE 2 62.86Complement C1q subcomponent subunit B (Mus musculus) 3.94⁻⁰³A6X935|ITIH4_MOUSE 4 301.36 Inter α-trypsin inhibitor, heavy chain 4(Mus musculus) 3.94⁻⁰³ E7D4P4|E7D4P4_MERUN 9 1172.7 Apolipoprotein E(Meriones unguiculatus) 3.78⁻⁰³ P97298|PEDF_MOUSE 5 420.63 Pigmentepithelium-derived factor (Mus musculus) 3.31⁻⁰³ P47878|IBP3_MOUSE 5520.36 Insulin-like growth factor-binding protein 3 (Mus musculus)3.10⁻⁰³ P46412|GPX3_MOUSE 3 275.46 Glutathione peroxidase 3 (Musmusculus) 2.99⁻⁰³ Q88H35|CO88_MOUSE 4 586.84 Complement component C8 βchain (Mus musculus) 2.99⁻⁰³ Q64118|A1AT_MERUN 3 183.98 α-1-antitrypsin(Meriones unguiculotus) 2.89⁻⁰³ Q06890|CLUS_MOUSE 5 427.41 Clusterin(Mus musculus) 1.91⁻⁰³ P70389|ALS_MOUSE 3 471.58 Insulin-like growthfactor-binding protein complex acid labile subunit 1.88⁻⁰³ (Musmusculus) P35441|TSP1_MOUSE 8 853.9 Thrombospondin-1 (Mus musculus)1.81⁻⁰³ P68033|ACTC_MOUSE 2 978.66 Actin, α cardiac muscle 1 (Musmusculus) 1.64⁻⁰³ Q00724|RET4_MOUSE 4 335.66 Retinol-binding protein 4(Mus musculus) 1.63⁻⁰³ P26262|KLKB1_MOUSE 5 625.92 Plasma kallikrein(Mus musculus) 1.52⁻⁰³ Q61704|ITIH3_MOUSE 4 422.55 Inter-α-trypsininhibitor heavy chain H3 (Mus-musculus) 1.52⁻⁰³ P19221|THRB_MOUSE 7587.54 Prothrombin (Mus musculus) 1.45⁻⁰³ P33434|MMP2_MOUSE 3 283.7 72kDa type IV collagenase (Mus musculus) 1.42⁻⁰³ Q9JHH6|CBPB2_MOUSE 3367.6 Carboxypeptidase B2 (Mus musculus) 1.38⁻⁰³ P32261|ANT3_MOUSE 2306.33 Antithrombin-III (Mus musculus) 1.30⁻⁰³ nLs.2.1.2.t03443-RA 3366.35 Hypothetical protein, Bm1_50630 homolog (Litomosoides 1.18⁻⁰³sigmodontis) nLs.2.1.2.t01366-RA 2 269.78 Transthyretin-like protein,partial (Litomosoides sigmodontis) 9.56⁻⁰⁴ P11680|PROP_MOUSE 2 36.41Properdin (Mus musculus) 9.27⁻⁰⁴ Q61646|HPT_MOUSE 3 236.91 Haptoglobin(Mus musculus) 8.65⁻⁰⁴ Q9JM99|PRG4_MOUSE 5 683.96 Proteoglycan 4 (Musmusculus) 8.57⁻⁰⁴ Q921I1|TRFE_MOUSE 4 728.55 Serotransferrin (Musmusculus) 8.09⁻⁰⁴ P28798|GRN_MOUSE 2 300.7 Granulins (Mus musculus)7.86⁻⁰⁴ P26928|HGFL_MOUSE 4 375.93 Hepatocyte growth factor-like protein(Mus musculus) 7.43⁻⁰⁴ Q9UN5|CBPN_MOUSE 3 219.53 Carboxypeptidase Ncatalytic chain (Mus musculus) 7.01⁻⁰⁴ P47879|IBP4_MOUSE 2 211.08Insulin-like growth factar-binding protein 4 (Mus musculus) 6.96⁻⁰⁴Q07968|F136_MOUSE 3 410.66 Coagulation factor XIII B chain (Musmusculus) 6.89⁻⁰⁴ Q9D6DD|ICA_MOUSE 6 787.37 Inhibitor of carbonicanhydrase (Mus musculus) 5.99⁻⁰⁴ P97290|IC1_MOUSE 4 387.11 Plasmaprotease C1 inhibitor (Mus musculus) 5.94⁻⁰⁴ Q8K182|CO8A_MOUSE 2 332.91Complement component C8 α chain (Mus musculus) 5.78⁻⁰⁴ O70362|PHLD_MOUSE2 100.55 Phosphatidylinositol-glycan-specific phospholipase D (Musmusculus) 4.92⁻⁰⁴ P01872|IGHM_MOUSE 2 174.92 Ig μ chain C regionsecreted form (Mus musculus) 4.48⁻⁰⁴ P06684|COS_MOUSE 2 338.69Complement CS (Mus musculus) 4.07⁻⁰⁴ Q8K0D2|HABP2_MOUSE 2 60.74Hyaluronan-binding protein 2 (Mus musculus) 3.53⁻⁰⁴ nLs.2.1.2.t01870-RA2 196.87 ML domain-containing protein (Litomosoides sigmodontis) 3.44⁻⁰⁴nLs.2.1.2.t01365-RA 2 188.68 Transthyretin-like protein, partial(Litomosoides sigmodontis) 2.74⁻⁰⁴ P28665|MUG1_MOUSE 3 312.94Murinoglobulin-1 (Mus musculus) 2.05⁻⁰⁴ Q08879|FBLN1_MOUSE 2 305.68Fibulin-1 (Mus musculus) 1.90⁻⁰⁴ Q8CG16|C1RA_MOUSE 2 102.86 ComplementC1r-A subcomponent (Mus musculus) 1.04⁻⁰⁴ IBAQ, intensity-based absolutequantification; AMBP, α-1-microglobulin/bikunin precursor.

TABLE 7 Putative surface-associated proteins exhibiting >50-foldenrichment in biotin-labelled adult worm whole body extracts relative tounlabelled contols Peptides Parasite used for Confidence Presence stageTreatment quantification score Fold-difference Locus tag Annotation inESP AM SDS 4 316.19 1,769.5 nLs_09715 Major sperm protein Yes SDS 2249.88 341.7 nLs_01747 Filarial antigen RAL-2 Yes SDS 6 873.95 62.2nLs_06907 Adenylate kinase isoenzyme 1 No PBS 4 172.54 50.6 nLs_09625Transthyretin-like protein 5 Yes GAF Urea 2 306.56 430.9 nLs_02969Cysteine protease inhibitor-2 Yes Urea 2 180.49 149.4 nLs_08458 Filarialantigen Ov16 Yes Urea 2 302.26 65.2 nLs_09625 Transthyretin-like protein5 Yes OG 2 233.80 60,617.8 nLs_09890 Purine nucleoside phosphorylase YesOG 2 183.22 336.5 nLs_00852 Proliferating cell nuctear antigen domainprotein No OG 2 224.62 271.9 nLs_04749 60S ribosomal protein L18 No OG 2191.67 168.3 nLs_01364 Transthyretin-lite protein, partial Yes OG 3194.25 156.3 nLs_02023 Tetratricopeptide-repeat domain protein Yes OG 2159.32 139.9 nLs_02001 KH domain-containing protein Yes OG 2 188.56118.1 nLs_08084 Type 1 inositol-trisphosphate 5-phosphatase Yes OG 3367.83 79.9 nLs_02969 Cysteine protease ihibitor-2 Yes OG 3 367.26 66.6nLs_02463 FKBP-type peptidyl-prolyl cis-trans isomerase Yes OG 3 220.0065.6 nLs_00523 KH domain centaining protein Yes OG 3 376.34 59.0nLs_3910 Wolbachia surface protein No OG 2 258.28 52.2 nLs_05241Tetratricopeptide-repeat domain protein No SDS 2 50.67 1,059.7 nLs_07759Cyclophlin Ovcyp-2 homologue Yes SDS 6 306.80 328.9 nLs_08458 Filarialantigen Ov16 Yes SDS 7 488.42 304.1 nLs_01747 Filarial antigen RAL-2 YesSDS 4 156.74 262.8 nLs_05279 HSP20/α-crystallin family protein No SDS 2144.16 242.7 nLs_08696 Lysozyme protein 8, partial Yes SDS 2 87.95 235.6nLs_09890 Purine nucleoside phosphorylase Yes SDS 7 432.37 216.4nLs_09625 Transthyretin-like protein 5 Yes SDS 2 82.06 202.7 nLs_2001 KMdomain-containing protein Yes SDS 6 332.26 193.2 nLs_08148 Papilin YesSDS 2 122.31 162.4 nLs_06907 Adenylate kinase isoenzyme 1 Yes SDS 244.96 162.1 nLs_00117 L-lactate dehyrogenase Yes SOS 3 344.30 148.4nLs_05914 Pyruvate dehydrogenase E1 component, α-subunit Yes SDS 2 77.92115.1 nLs_09750 Transthyretin-like protein 45 Yes SDS 7 329.76 86.5nLs_03034 p27 heat shock protein homologue Yes SDS 5 166.70 82.3nLs_08836 von Willebrand factor type-d domain protein Yes SDS 3 138.7071.7 nLs_03328 Myosin No SDS 2 89.16 67.5 nLs_01364 Transthyretin-likeprotein, partial Yes SDS 6 560.89 52.8 nLs_08415 Enolase Yes PBS 5631.96 111.4 nLs_02378 Aldo/keta reductase family protein No PBS 2309.24 104.0 nLs_03070 Atypical RIO/RIO2 protein kinase Yes PBS 3 572.5993.5 nLs_00473 Aldehyde dehydrogenase 11 Yes PBS 6 1058.57 89.2nLs_06488 Acid phosphatase Yes PBS 9 1570.87 68.9 nLs_01747 Filarialantigen RAL-2 Yes PBS 4 647.70 62.5 nLs_03174 Nematode secreted protein22U Yes ESP, ecretory-secretory products; AM, adult male; OG, octyl β-Dglucopyranoside; GAF, gravid adult female; FKBP, FK506-binding protein;HSP, heat-shock protein.

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Sequence Information and Comparisons

MSPFILLALLINAPANCRPDNGISRSRDASSA

SDTVSGSSARPSSQFLPSSSQRQSLALTSGAVE KERKSLTS

SDDVAIGSGSTTAAHRSTAFGVEKFKGGSASSS LSPRIGNALISGSL

SVDTSISSSSSNATLRVMSPSV EIGGSSGGTSSHRTAKQDSYEANHNIPAYPRLS RGEELE

RKGSVVEERHSSLPAAQGNKATAITKE

GEKSSYGSVI ELESPIAASSDEGSVIALDSDGNDGSSTRSTMTSERRLTSGSGDTMSMQKPKHSSIRGRTDPIRSSSSAS TAHIQQPTNKQYLGTQRYPGRTGP

Above is domain organisation of the ShK domain-containing proteinnLs_04059 from Litomosoides sigmodontis. Linear representation of theamino-acid sequence (SEQ ID NO: 1), showing the signal peptide (initalics), six ShK toxin-like domains (open rectangles) containing sixcysteine residues each (highlighted), and a predicted propeptidecleavage site (underlined). Domain six at the C-terminus is unique incontaining two lysyltyrosine dyads (bold).

Diro. MGKYKGEIXCCGTGTSCKNICLKFSEFACNSCAKTCGILQSSGRSGVCYDKDPDCSDDVC Lito.---------------MSPFILLALLINAPANCRPDNGISRSRDASSACYDKDPDCSSDIC                .  * * :   *  .*    ** :*   *..*********.*:* Diro.RNYPYTAKERCPKYCGLCHDSSLRSGNRLSSGLSSSYQQSSSSSLPSLKSGITGSTIIKK Lito.KNYPYTAKERCPKFCGLCSDTVSGSSARPSSQFLPSSSQRQSL---------ALTSGAVE:************:**** *:   *. * ** :  * .* .*          : ::   : Diro.DERKSSLPCIDKDSDCNMEICRNFPYTAKERCAKTCGLCSGETSSS-GIT--SGHHTIAG Lito.KERKSLTSCTDKDSDCTAEICRNYPFTARERCAKTCGRCSDDVAIGSGSTTAAHRSTAFG.****   * ******. *****:*:**:******** ** :.: . * * :  : *  * Diro.IDKSRGGT-TSLLSSARGNEPFSSGLCFDKKLDCRKEICRDFPFTAKEECAKTCGFCSSD Lito.VEKFKGGSASSSLSPRIGNALISGSLCFDRKFDCSREICRDFPFTARQECAKTCGFCSVD::* :**: :* ** * **  :*..****:*:** :**********::********** * Diro.KGMSSSSSSGTAFGTMSPSRHAS--------IRINERDGITGIRSTSPHSILSKEKDLEC Lito.TSISSSSSNA-TLRVMSPSVEIGGSSGGTSSHRTAKQDSYEANHNIPAYPRLSRGEELEC..:*****.. :: .**** . .         *  ::*.  . :.   :  **: ::*** Diro.TDLNTDCTQQICKDYPYTAKERCAKTCGFCRREMTEGDKTSVGGRHSSFTDKQRSRISEL Lito.VDVNIDCTQQTCKDYPFTARERCAKTCGFCRKGSVVEE------RHSSL-----------.*:* ***** *****:**:***********:  .  :      ****: Diro.DSRDSSLRGIKSSPTTEDCRDEDSQCSEKSCLDRPYTARTKCAKTCGFCGS------TVD Lito.----PAAQGNKATAITKECKDEDSQCSERSCLEHPYKASRKCAKTCGFCGEKSSYGSVIE     : :* *::  *::*:********:***::**.*  **********.      .:: Diro.LEPPLVDSLDKGNIITLDDDVTT----RSTATFDRHSTSGIGTP--TQSSRHLSVGSRTD Lito.LESPIAASSDEGSVIALDSDGNDGSSTRSTMTSERRLTSGSGDTMSMQKPKHSSIRGRTD** *:. * *:*.:*:**.* .     *** * :*: *** *     *. :* *: .*** Diro.SSRK--PSLSTHIQQPTRRPFQGVLGRYPGRTGLCADENAYCQKEDCYKYPRFGQRYCEK Lito.PIRSSSSASTAHIQQPTNKQYLGT-QRYPGRTGPCTDANQLCEKADCYKYPNFSQKYCEK  *.   : ::******.: : *.  ******* *:* *  *:* ******.*.*:**** Diro. TCNYCLito. TCNYC

Above is a sequence alignment comparison between ShK domain-containingproteins nLs.2.1.2.t04059-RA (from Litomosoides sigmodontis) of SEQ IDNO: 1 and nDi.2.2.2.t03402-RA (from Dirofilaria immitis) of SEQ ID NO:2.

While overall identity between the two sequences is only 52.8%, it canbe seen that the identity shared between the ShK domains (highlighted)of these two filarial nematode proteins is considerably higher.

The invention claimed is:
 1. A polypeptide comprising: at least one ShKdomain of L. sigmoidontis protein nLs_04059 according to SEQ ID NO:1;and/or at least one polypeptide sharing at least 70 percent identitywith said at least one ShK domain, wherein the at least one polypeptideretains 6 cysteine residues with characteristic spacing of an ShKdomain; wherein the polypeptide is artificial.
 2. The polypeptideaccording to claim 1, wherein the polypeptide is a chimeric polypeptide.3. The polypeptide according to claim 1, wherein the at least onepolypeptide sharing at least 70 percent identity with said ShK domain isfrom a filarial nematode selected from group consisting of: L.sigmodontis, D. immitis, Wuchereria bancrofti, Brugia malayi, Brugiatimori, Onchocerca volvulus, Loa loa.
 4. A nucleic acid molecule,comprising: at least one nucleic acid sequence encoding at least onepolypeptide comprising at least one ShK domain of L. sigmoidontisprotein nLs_04059 according to SEQ ID NO:1; and/or at least one nucleicacid encoding at least one polypeptide sharing at least 70 percentidentity with said at least one Shk domain wherein the at least onepolypeptide retains 6 cysteine residues with characteristic spacing ofan ShK domain; wherein the nucleic acid molecule is artificial.
 5. Anexpression vector comprising the nucleic acid molecule according toclaim 4, optionally wherein said expression vector is for expression inE. coli.
 6. The nucleic acid according to claim 4, wherein the at leastone polypeptide sharing at least 70 percent identity with said at leastone ShK domain is from a filarial nematode selected from groupconsisting of: L. sigmodontis, D. immitis, Wuchereria bancrofti, Brugiamalayi, Brugia timori, Onchocerca volvulus, Loa loa.
 7. A method oftreating or preventing a filarial nematode infection, comprising thesteps of: providing to a subject a therapeutically effective amount ofat least one polypeptide comprising at least one ShK domain of L.sigmoidontis protein nLs_04059 according to SEQ ID NO:1 and/or at leastone polypeptide sharing at least 70 percent identity with at least oneShk domain wherein the at least one polypeptide retains 6 cysteineresidues with characteristic spacing of an ShK domain.
 8. The methodaccording to claim 7, wherein the subject is a human or an animal. 9.The method according to claim 7, wherein the filarial nematode infectionincludes a disease selected from the group consisting of: lymphaticfilariasis, onchocerciasis, and loiasis.
 10. The method according toclaim 7, wherein the subject is an animal and the filarial nematodeinfection is heartworm.
 11. The method according to claim 7, wherein theat least one polypeptide sharing at least 70 percent identity with atleast one Shk domain of a filarial nematode protein is from a filarialnematode selected from a group consisting of: L. sigmodontis, D.immitis, Wuchereria bancrofti, Brugia malayi, Brugia timori, Onchocercavolvulus, and Loa loa.
 12. The method according to claim 7, wherein theat least one ShK domain and/or the at least one polypeptide sharing atleast 70 percent identity with said at least one Shk domain isformulated as a pharmaceutical composition.
 13. The method according toclaim 10, further comprising the step of: administering a nucleic acidmolecule encoding the therapeutically effective amount of the at leastone polypeptide.
 14. The method according to claim 10, wherein the atleast one polypeptide comprises a plurality of the same or different ShKdomains.
 15. The method according to claim 10, wherein the at least onepolypeptide is a chimeric polypeptide, optionally wherein the at leastone polypeptide further comprises an artificial spacer separating theShK domains.
 16. The method according to claim 10, wherein the at leastone polypeptide further comprises an additional vaccine antigen.